Development of a temperature-switch PCR-based SNP typing method for Mycobacterium ulcerans

Abstract

Mycobacterium ulcerans (M. ulcerans), the causative agent of the devastating skin disease Buruli ulcer (BU), is characterized by an extremely low level of genetic diversity. Recently, we have reported the first discrimination of closely related M. ulcerans variants in the BU endemic Densu River Valley of Ghana. In the study real-time PCR-based single nucleotide polymorphism (SNP) typing at 89 predefined loci revealed the presence of ten M. ulcerans haplotypes circulating in the BU endemic region. Here we describe the development of temperature-switch PCR (TSP) assays that allow distinguishing these haplotypes by conventional agarose gel-based analysis of the PCR products. After validation of the accuracy of typing results, the TSP assays were successfully established in a reference laboratory in Ghana. Development of the cost-effective and rapid TSP-based genetic fingerprinting method will thus allow investigating the spread of M. ulcerans clones by regular genetic monitoring in BU endemic countries.

Figure 1. Selection of TSP SNP typing assays.

A Linearized phylogenetic tree of the ten M. ulcerans haplotypes (HT1–10) detected in the Densu River Valley of Ghana (MEGA software version 4.1 (beta), scale: number of differences at the 89 SNP loci tested). B Schematic overview of reference (0) and SNP (1) alleles present in the sequence of the ten M. ulcerans haplotypes, which can be identified by TSP assays 1, 3, 4, 6, 8, 9, 15, 16, 17 and 18.

Figure 2. Schematic illustration of TSP assay performance.

NAS and LS primer locations relative to the M. ulcerans DNA sequence surrounding a SNP locus are shown for the different PCR reaction phases. A Initial PCR conditions enable an amplification of the larger LS PCR product by applying an annealing temperature (Ta) of 58°C. B Reduction of Ta to 45°C facilitates a possible incorporation of the NAS primer into the enriched LS PCR product. C Competitive amplification of the larger LS and the smaller NAS PCR products are ensured by an increase of Ta to 53°C.

Figure 3. Optimization of TSP SNP typing assays.

TSP endpoint detection by analysis of PCR product sizes on ethidium bromide-stained agarose gels for haplotypes 1–10 (lanes 1–10) at TSP assay locus 6. TSPs were performed using: A a four-primer system including both NAS and LS forward and reverse primers as well as a three-primer system with only one NAS primer and different NAS∶LS primer ratios of B 2.5∶1, C 5∶1, D 7.5∶1 and E 10∶1.

Figure 4. Application of TSP assay 16 in two different research laboratories.

Comparison of TSP endpoint detection by analysis of PCR product sizes on ethidium bromide-stained agarose gels in two different laboratories in Basel, Switzerland and Accra, Ghana. PCR products are shown for haplotypes 1–10 (lanes 1–10).