A new cost-effective and fast direct PCR protocol for insects based on PBS buffer.

Area of Science:

• Entomology
• Molecular Biology
• Genetics

Background:

• DNA barcoding is crucial for insect biodiversity assessment.
• Traditional DNA extraction methods can lead to significant DNA loss.
• Existing direct PCR techniques have limitations in success rates and sample reusability.

Purpose of the Study:

• To develop a fast and effective direct PCR workflow for universal insect species identification.
• To improve DNA yield and enable further genetic studies from insect samples.

Main Methods:

• A novel 2-minute sample preparation in PBS-buffer for direct PCR.
• Testing the protocol across various insect orders and specimen preservation types.
• Evaluating success rates for DNA amplification and sequencing quality.

Main Results:

• Achieved 100% amplification success in six insect orders (Mantodea, Phasmatodea, Neuroptera, Odonata, Blattodea, Orthoptera).
• High success rates (75-97.3%) for Lepidoptera, Coleoptera, Diptera, Hemiptera, and Hymenoptera.
• Demonstrated sensitivity to small sample amounts and effectiveness on diverse sample types, including degraded specimens.

Conclusions:

• The developed direct PCR workflow offers a low-cost, simple, and highly effective method for insect species identification.
• The protocol is beneficial for insect taxonomy and ecological studies, especially with degraded samples.
• The pre-PCR solution allows for subsequent DNA extraction, enhancing sample utility.