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Development of a loop-mediated isothermal amplification detection assay for Dictyocaulus viviparus (Bloch, 1782)
Sirapat Nak-On1,2, Paul Campbell2, Maha Mansour Shalaby2,3,4
1Applied Parasitology Research Laboratory, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand.
Abstract:
The bovine lungworm, Dictyocaulus viviparus (Bloch, 1782), is highly pathogenic and disease outbreaks can be difficult to predict and manage. Rapid and accurate diagnosis is vital, but without a sensitive diagnostic test this remains challenging in clinical practice. High performance molecular detection tools are therefore required to improve the diagnosis of this parasite and promote the implementation of strategic control measures. Loop-mediated isothermal amplification (LAMP), a rapid DNA assay, offers potential for field-based detection. Here we report a novel LAMP assay (DviLAMP), that was designed to target the D. viviparus internal transcribed spacer 2 (ITS2) ribosomal DNA region. Firstly, genomic DNA was extracted from a single D. viviparus L1 larva to amplify and clone the ITS2 into the recombinant plasmid (DviITS2). The DviLAMP successfully detected the target, with results shown by gel electrophoresis and real-time analysis, in addition to point-of-care amenable end-point detection: colorimetry and lateral flow dipstick (LFD). Analytical sensitivity can detect 0.5 ng DviITS2 following 45 min of incubation at 64°C, increasing to just 1 pg following 90 min of incubation. Using the same primers, other nematodes of cattle, Ostertagia ostertagi and Cooperia oncophora, were also detectable both by gel electrophoresis and real-time. However, when FITC and biotin tagged primers were incorporated to adapt the DviLAMP to LFD end-point detection, the LFD showed specific detection of D. viviparus. Further development of DviLAMP as a point-of-care test could significantly improve the sensitivity of lungworm diagnosis in the field.
Insights
A new Loop-mediated isothermal amplification (LAMP) assay accurately detects the bovine lungworm, Dictyocaulus viviparus. This rapid molecular tool shows promise for sensitive, point-of-care diagnosis in the field.
Area of Science:
- Veterinary Parasitology
- Molecular Diagnostics
- Molecular Biology
Background:
- Bovine lungworm (Dictyocaulus viviparus) causes significant disease, with outbreaks challenging to predict and manage.
- Accurate and rapid diagnosis is crucial for effective control but is hindered by a lack of sensitive diagnostic tests.
- High-performance molecular detection tools are needed to improve Dictyocaulus viviparus diagnosis and control strategies.
Purpose of the Study:
- To develop a novel, rapid, and sensitive Loop-mediated isothermal amplification (LAMP) assay for the molecular detection of Dictyocaulus viviparus.
- To evaluate the diagnostic performance of the LAMP assay, including its analytical sensitivity and specificity.
- To explore the potential of the LAMP assay for point-of-care diagnostics.
Main Methods:
- A novel LAMP assay (DviLAMP) was designed targeting the Dictyocaulus viviparus internal transcribed spacer 2 (ITS2) ribosomal DNA region.
- Genomic DNA from Dictyocaulus viviparus L1 larvae was used to amplify and clone the ITS2 region.
- The DviLAMP assay was validated using gel electrophoresis, real-time analysis, colorimetry, and lateral flow dipstick (LFD) detection.
Main Results:
- The DviLAMP assay successfully detected the target Dictyocaulus viviparus ITS2 DNA.
- Analytical sensitivity reached 0.5 ng DNA in 45 minutes and 1 pg DNA in 90 minutes.
- While initial tests detected other cattle nematodes, LFD adaptation with tagged primers demonstrated specific detection of Dictyocaulus viviparus.
Conclusions:
- The developed DviLAMP assay is a sensitive molecular tool for Dictyocaulus viviparus detection.
- The assay's adaptability for point-of-care testing, such as with LFD, holds significant potential for improved field diagnosis.
- Further development could enhance the sensitivity and accessibility of lungworm diagnosis in veterinary practice.
