Key Points
- PCR assays for species identification and insecticide resistance in Anopheles gambiae were run using a portable laboratory setup.
- The protocols included multiplex PCR for species ID and detection of kdr-East and kdr-West resistance mutations.
- This demonstration shows how validated assays can be implemented in decentralised settings without full laboratory infrastructure.
We recently tested two PCR protocols developed by the CDC for Anopheles mosquito analysis, using Bento Lab. Our goal was to demonstrate how these assays — originally designed for use in standard lab environments — could be successfully run on portable equipment, opening the door to field-based molecular surveillance workflows.
🎥 Watch the full demonstration on YouTube
A Field-Compatible Protocol
Molecular assays are increasingly important in malaria vector surveillance, but standard PCR protocols often assume access to a fully equipped laboratory. To support more decentralised approaches — such as field studies and regional surveillance activities — we tested whether these CDC protocols could be carried out using only Bento Lab.
The assays were performed on our equipment, which is already in use by researchers working on this protocol. While we did not develop or validate the assays ourselves, this demonstration was intended to show how the existing validated methods can be implemented in portable laboratory environments.
This approach offers promising potential for rapid, on-site analysis in settings such as:
- Field research stations
- Surveillance hubs
- Insecticide resistance monitoring programs
Overview of the Protocols
The video demonstration walks through two CDC-developed assays using Anopheles gambiae mosquitoes supplied by the CDC, with reagents and controls from the Malaria Research and Reference Reagent Resource Center (MR4, BEI Resources):
Protocol 1:
Species identification within the Anopheles gambiae complex using a standard multiplex PCR.
This assay distinguishes between six morphologically similar mosquito species using a set of species-specific primers. It’s a cornerstone protocol for entomological studies and malaria transmission mapping.
Protocol 2:
Detection of knockdown resistance (kdr) mutations, which are associated with pyrethroid insecticide resistance.
Two PCRs are used to detect kdr-East and kdr-West variants — common mutations that signal resistance pressures in East and West African mosquito populations, respectively.
Summary of the Demonstration Workflow
The video provides a complete walkthrough of the laboratory procedures, from DNA extraction through PCR amplification to gel electrophoresis.
Sample Preparation and DNA Extraction
The workflow begins with mosquito sample preparation. Two DNA extraction methods are demonstrated: the HOTShot protocol and a rapid Dipstick-based method. Both are suited to low-resource environments and were successfully used with Bento Lab.
PCR Amplification
PCR reactions for both protocols are prepared and run using Bento Lab’s built-in thermal cycler. Protocol 1 is used first to identify species within the Anopheles gambiae complex. This is followed by Protocol 2, targeting kdr resistance alleles.
Gel Electrophoresis and Results
The amplified products are visualised using Bento Lab’s electrophoresis module. The video shows clear results consistent with expected fragment sizes for both assays.
The demonstration concludes with a summary of the results and remarks on the practical workflow.
Implications for Field-Based Molecular Analysis
This demonstration confirms that CDC-validated assays for malaria vector identification and insecticide resistance can be implemented on a portable laboratory platform. For researchers working in field settings, regional labs, or surveillance programs, this enables access to high-quality molecular diagnostics without relying on centralised infrastructure.
Such portability expands the scope of on-site analysis and contributes to more timely, locally-driven public health responses.
