Rapid on-site universal vertebrate species identification via multi-barcode nanopore sequencing

Area of Science:

• Conservation genetics
• Forensic science
• Molecular biology

Background:

• Illegal wildlife trade (IWT) poses significant threats to biodiversity and public health.
• Current DNA-based species identification methods are time-consuming, costly, and require laboratory facilities.
• There is a critical need for rapid, on-site identification tools to combat IWT.

Purpose of the Study:

• To develop and validate VeRIF-ID (Vertebrate Rapid In-Field Identification via DNA), a novel method for rapid, on-site species identification.
• To enable accurate identification of trafficked wildlife species, even from processed or damaged samples.
• To provide a cost-effective and portable solution for species identification in the field.

Main Methods:

• Simultaneous on-site nanopore sequencing of four mitochondrial DNA barcodes using specifically designed primers.
• Simplified DNA extraction, PCR, and library preparation for rapid analysis (<3 hours per sample).
• Validation using diverse vertebrate species, including testing on Traditional East Asian Medicine samples and seized bushmeat.

Main Results:

• VeRIF-ID achieved species-level identification in 91% of 83 tested species, with genus/tribe-level identification for the remainder.
• Identified critically endangered species (saiga antelope, black rhinoceros) in Traditional East Asian Medicine samples.
• Successfully identified mammal and fish species in seized bushmeat samples at an airport customs zone within 6 hours.

Conclusions:

• VeRIF-ID offers a rapid, cost-effective (<$48/sample), and portable solution for on-site species identification.
• The method is effective for identifying species from processed and damaged DNA, crucial for combating illegal wildlife trade.
• VeRIF-ID has the potential to democratize species identification and enhance enforcement efforts against wildlife crime and zoonotic disease transmission.