Development of a qPCR assay to identify and differentiate insect-associated strains of the Serratia marcescens complex

Abstract

The Serratia marcescens complex contains important opportunistic pathogens of humans and vertebrate animals, as well as insects and other invertebrates. To date, the methods used for the identification of species within the genus Serratia, including PCR assays, have poor discriminatory power and may require further molecular typing or genomic sequence analysis to determine clinical relevance. We developed a duplex TaqMan probe-based quantitative real-time PCR (qPCR) assay targeting the chiP gene, which is involved in chitin degradation and transport, and the ureD gene, which is involved in urease production. This allowed us to simultaneously identify all members of the S. marcescens complex (chiP positive) and differentiate those most likely to act as insect pathogens (chiP and ureD positive). We applied our assay to identify potentially entomopathogenic members of the S. marcescens complex in the context of a conservation program for the critically endangered insect Dryococelus australis and found it to be a useful aid for rapid and accurate detection of infection with S. marcescens complex strains in insects and determination of their clinical relevance. By targeting 2 genes likely to be virulence factors, this assay may also be of use for research investigating the pathogenesis of entomopathogenic Serratia spp.

Keywords: Dryococelus australis; chitinase; entomopathogen; multiplex qPCR; urease.

Figure 1.

Primer sets aligned to the corresponding gene sequences from reference genomes for Serratia spp. and non-Serratia spp. containing the (A) chiP and (B) ureD genes. Multiple alignments of the primer sets with the reference sequences were created and visualized using Geneious Prime v.2021.1.1 (Biomatters) with the Multiple Align tool and default settings for a Geneious alignment.

Figure 2.

Duplex qPCR assay for the Serratia chiP and ureD targets with standard curves for S. ureilytica AM923 gDNA. Amplification plots for the Serratia (A) chiP and (B) ureD targets over a dynamic range of 10⁷–10³ copies/reaction (curves 1–5) and negative control (curve 6). Standard curves of the assay for the Serratia (C) chiP and (D) ureD targets with regression equations and regression coefficients (r²).