Firebird's PCR-based 20-plex assay for detecting enteropathogen and antimicrobial resistant genes in environmental samples

This multiplexed PCR assay leverages both AEGIS and SAMRS primer technologies for amplification specificity and next-generation sequencing for amplicon identification.

Designed to minimize both the primer dimer issues commonly faced in standard multiplex PCR assays and amplification bias common when analyzing complex samples of unknown composition, the assay enables detection of 18 enteropathogens from bacteria, viruses, protozoa, and helminths, as well as two antimicrobial resistant genes. 

Enteric Pathogens & Antimicrobial Resistance Genes Detected in the 20-Plex Assay:

How it works:

• (a) Standard DNA primers in a PCR reaction can dimerize and cross-prime, consuming available primers and dNTPs.
• (b) strategically inserted SAMRS bases minimize primer dimer formation.
• (c) PCR amplification of all 20 targets (40 primers) using standard DNA or SAMRS primers shows standard DNA primer dimerization; SAMRS primers show no visible dimerization.
• (d) The first round of amplification in the SAMRS-AEGIS 20-plex reaction uses primers containing SAMRS bases in the target binding region, and the AEGIS P base in an overhang tag region. These primers amplify targets of interest while minimizing primer dimerization and non-specific amplification. The corresponding Z triphosphate (dZTP) is included during amplification.
• (e) The second amplification reaction uses primers containing the AEGIS P base to bind to the tag region added during the first round. Here, the AEGIS bases prevent non-specific amplification due to their lack of pairing with standard DNA bases.
• AEGIS barcode primers thus allow for highly specific amplification and enrichment of products formed by SAMRS-AEGIS primers during the first round of PCR. After a second PCR amplification samples are purified, pooled, and prepared for next-generation sequencing.

Adaptable to different sequencing platforms, the assay offers flexibility in the selection of barcode and adapter sequences and supports integration with user-selected sequencing workflows.

Note: Users are responsible for obtaining all reagents and consumables required for barcode incorporation, library preparation, sequencing, and downstream bioinformatic analysis that are not included with the assay kit. Find explicit instructions and recommendations on using product in the user guide provided. Please inquire for recommended materials.

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