Detection and enumeration of Lak megaphages in microbiome samples by endpoint and quantitative PCR

Abstract

Lak megaphages are prevalent across diverse gut microbiomes and may potentially impact animal and human health through lysis of Prevotella. Given their large genome size (up to 660 kbp), Lak megaphages are difficult to culture, and their identification relies on molecular techniques. Here, we present optimized protocols for identifying Lak phages in various microbiome samples, including procedures for DNA extraction, followed by detection and quantification of genes encoding Lak structural proteins using diagnostic endpoint and SYBR green-based quantitative PCR, respectively. For complete details on the use and execution of this protocol, please refer to Crisci et al., (2021).

Keywords: Health Sciences; Immunology; Microbiology; Molecular Biology.

Graphical abstract

Figure 1

Representative Lak phage major capsid PCR products visualised by agarose gel electrophoresis GR = 1 kb+ GeneRuler; + = Swine positive control; Lak MCP PCR amplicons from pig gastrointestinal samples = JE=Jejunum; IL = Ileum; CE = Caecum; PS = Proximal spiral; DS = Distal spiral. See Table 1 for primer details. Data relating to this figure has been published previously by Crisci et al. (2021).

Figure 2

Standard curves generated using Lak phage qPCR primers Cq = Cycle of quantification; R² = Coefficient of determination; RMSE = Root mean square error. Black dots represent technical replicates for each serial dilution of Lak major capsid amplicon (MCP: LakMC581-F/LakMC1053-R; MCP_660: LakHMC185-F/ LakHMC984-R): 1:10 starting at 5 ng. Primer efficiencies (E) calculated from slopes ((−1+10^−1/slope) × 100). (A) Lak MCP amplicons used as standards with LakMC683-F/LakMC803-R primers, E = 102.8%. (B) Lak MCP_660 variant amplicons used as standards with LakHMC224-F/LakHMC421-R primers, E = 110.1%. See Table 1 for primer details. Data relating to this figure has been published previously by Crisci et al. (2021).

Figure 3

Representative melt curves for Lak qPCR primer pairs RFU = relative fluorescence units using SYBR green. Single peak generated from 3 technical replicates indicates no nonspecific binding or secondary structures. (A) Representative result shown for Lak MCP primers (LakMC683-F/LakMC803-R). (B) Representative result shown for Lak MCP_660 primers (LakHMC224-F/LakHMC421-R). See Table 1 for primer details. A version of this figure has been published previously by Crisci et al. (2021).