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Development and validation of an arthropod maceration protocol for zoonotic pathogen detection in mosquitoes and
Genelle F Harrison1, Jessica L Scheirer2, Vanessa R Melanson3
1McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, H3A 1B1, Canada.
Abstract:
Arthropod-borne diseases remain a pressing international public health concern. While progress has been made in the rapid detection of arthropod-borne pathogens via quantitative real-time (qPCR), or even hand-held detection devices, a simple and robust maceration and nucleic acid extraction method is necessary to implement biosurveillance capabilities. In this study, a comparison of maceration techniques using five types of beads followed by nucleic acid extraction and detection were tested using two morphologically disparate arthropods, the Aedes aegypti mosquito and Xenopsylla spp. flea, to detect the zoonotic diseases dengue virus serotype-1 and Yersinia pestis. Post-maceration nucleic acid extraction was carried out using the 1-2-3 Platinum-Path-Sample-Purification (PPSP) kit followed by qPCR detection using the Joint Biological Agent Identification and Diagnostic System (JBAIDS). We found that the 5mm stainless steel beads added to the beads provided in the PPSP kit were successful in macerating the exoskeleton for both Ae. aegypti and Xenopsylla spp. Replicates in the maceration/extraction/detection protocol were increased in a stepwise fashion until a final 128 replicates were obtained. For dengue virus detection there was a 99% positivity rate and for Y. pestis detection there was a 95% positive detection rate. In the examination of both pathogens, there were no significant differences between qPCR instruments, days ran, time of day ran, or operators.
Insights
A new method using stainless steel beads effectively macerates arthropods for pathogen detection. This technique significantly improves the detection rates of dengue virus and Yersinia pestis, crucial for public health surveillance.
Area of Science:
- Public Health
- Vector-borne Diseases
- Molecular Diagnostics
Background:
- Arthropod-borne diseases pose a significant global health challenge.
- Current detection methods like quantitative real-time PCR (qPCR) require improved sample preparation.
- A simple, robust method for arthropod maceration and nucleic acid extraction is needed for effective biosurveillance.
Purpose of the Study:
- To develop and evaluate a simple, robust maceration and nucleic acid extraction method for arthropod-borne pathogen detection.
- To compare different bead types for efficient arthropod exoskeleton maceration.
- To assess the effectiveness of the optimized protocol for detecting dengue virus and Yersinia pestis.
Main Methods:
- Tested five types of beads for maceration of Aedes aegypti mosquitoes and Xenopsylla spp. fleas.
- Utilized the 1-2-3 Platinum-Path-Sample-Purification (PPSP) kit for nucleic acid extraction.
- Employed the Joint Biological Agent Identification and Diagnostic System (JBAIDS) for quantitative real-time PCR (qPCR) detection.
- Optimized the protocol by increasing replicates to 128 for enhanced sensitivity.
Main Results:
- 5mm stainless steel beads, combined with provided kit beads, effectively macerated both mosquito and flea exoskeletons.
- Achieved high detection rates: 99% for dengue virus serotype-1 and 95% for Yersinia pestis.
- No significant variations in detection results were observed across different qPCR instruments, days, times, or operators.
Conclusions:
- The developed maceration and nucleic acid extraction protocol using stainless steel beads is effective and robust for arthropod-borne pathogen detection.
- This method enhances biosurveillance capabilities by providing reliable detection of key zoonotic diseases.
- The protocol's consistency across various parameters ensures its suitability for widespread implementation.
