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Rapid detection of Bombyx mori nucleopolyhedrovirus (BmNPV) by loop-mediated isothermal amplification assay combined
Yang Zhou1, Jiege Wu2, Feng Lin3
1Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
Abstract:
The Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the domestic silkworm. The disease often breaks out in sericultural countries and due to its high infectivity; it is difficult to control, resulting in heavy economic loss. In order to develop a rapid, sensitive visual detection and simple-to-use novel technology for detection of BmNPV, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) method was described. In this study, a set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the BmNPV gp41 gene, and the LAMP for the detection of BmNPV was developed by isothermal amplification at 61 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min. The detection limit of LAMP-LFD was 0.2 pg DNA extracted from silkworm infected with BmNPV and was 100 times more sensitive than conventional PCR. No product was generated from silkworm infected with other viruses. Furthermore, we applied the technique to detect BmNPV in the hemolymph and feces at different intervals post infection (pi). In conclusion, the novel LAMP-LFD setup presented here is simple, rapid, reliable, and has the potential for future use in the detection of BmNPV.
Insights
A new Loop-Mediated Isothermal Amplification (LAMP) assay combined with a Lateral Flow Dipstick (LFD) method offers rapid, sensitive detection of Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworms.
Area of Science:
- Virology
- Molecular Biology
- Biotechnology
Background:
- Bombyx mori nucleopolyhedrovirus (BmNPV) causes significant economic losses in sericulture due to its high infectivity and difficulty in control.
- Existing detection methods for BmNPV may lack the required speed, sensitivity, or ease of use for effective disease management.
Purpose of the Study:
- To develop a novel, rapid, sensitive, and user-friendly diagnostic technology for the detection of BmNPV.
- To establish a Loop-Mediated Isothermal Amplification (LAMP) assay coupled with a Lateral Flow Dipstick (LFD) method for BmNPV identification.
Main Methods:
- Design of specific primers and an FITC-labeled DNA probe targeting six regions of the BmNPV gp41 gene.
- Isothermal amplification at 61°C for 45 minutes, followed by probe hybridization and detection using LFD within 10 minutes.
Main Results:
- The developed LAMP-LFD assay demonstrated a detection limit of 0.2 pg BmNPV DNA, showing 100-fold greater sensitivity than conventional PCR.
- The assay exhibited high specificity, producing no detectable product from silkworms infected with other viruses.
- The technique was successfully applied to detect BmNPV in hemolymph and feces at various post-infection intervals.
Conclusions:
- The novel LAMP-LFD method provides a simple, rapid, and reliable means for BmNPV detection.
- This technology holds significant potential for practical application in BmNPV diagnostics and disease control in silkworm farming.
