Highly efficient multiplex PCR of noninvasive DNA does not require pre-amplification.

Area of Science:

• Wildlife genetics
• Molecular ecology
• Conservation biology

Background:

• Effective wildlife monitoring relies on informative genetic markers and efficient analysis methods.
• Commercial genotyping kits are common for humans and domestic animals but scarce for wildlife.

Purpose of the Study:

• To develop a highly optimized, cost-effective multiplex PCR protocol for genotyping brown bear (Ursus arctos) faecal samples.
• To improve the success rate of genetic analysis in wildlife research.

Main Methods:

• Developed a multiplex PCR assay for 12 microsatellite loci and a sex determination locus.
• Applied the protocol to 1053 brown bear faecal samples from the Dinaric population.
• Compared performance against a multiplex pre-amplification strategy on a subset of samples.

Main Results:

• Achieved an 88% genotyping success rate with the new multiplex PCR protocol, outperforming a previous 78.4% success rate.
• Direct comparison showed no advantage of multiplex pre-amplification for reasonably fresh non-invasive samples.
• The new protocol offers a high number of multiplexed loci, comparable to commercial kits.

Conclusions:

• The developed multiplex PCR is a reliable and cost-effective tool for brown bear genetic analysis from faecal samples.
• This method enhances the feasibility of molecular genetics in wildlife monitoring and research.
• Pre-amplification strategies are not justified for analyzing fresh non-invasive samples due to cost and workload.