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.

Whole Genome Comparisons Suggest Random Distribution of Mycobacterium ulcerans Genotypes in a Buruli Ulcer Endemic Region of Ghana

“…ulcerans is the close correlation between genotype and the geographic origin of a strain, but its restricted genetic diversity has limited the application of traditional molecular epidemiological methods such as VNTR-typing to discriminate between isolates at the village or even regional scales. The advent of low cost genomics has opened up new possibilities to explore and track the movement and spread of this pathogen within communities [ 29 , 30 ].…”

Late Onset of the Serological Response against the 18 kDa Small Heat Shock Protein of Mycobacterium ulcerans in Children

“…The results of a case-control study carried out in a BU endemic region of south-eastern Australia indicated reduced odds of having BU for individuals who frequently used insect repellent and increased odds for those who were bitten by mosquitoes [21] . In African BU endemic settings, the highly focal transmission of M. ulcerans haplotypes [12] , [22] , [23] , as well as the distribution pattern of BU lesions on the body [11] , speak against an exclusive role of mosquito vectors in transmission. Here we observed in children <5 years frequent sero-conversion for the MSP-1 antigen of the mosquito-transmitted malaria parasites in the absence of an IgG response against the M. ulcerans 18 kDa shsp.…”

Genetic Diversity of PCR-Positive, Culture-Negative and Culture-Positive Mycobacterium ulcerans Isolated from Buruli Ulcer Patients in Ghana

“…SNP typing has shown greater strain discrimination among M. ulcerans than VNTR typing [10] , [11] but has not yet been successfully used on patient samples or environmental samples. Results presented here support the importance of developing finer molecular tools for identification of M. ulcerans as well as the importance of including a 25°C incubation temperature for improving isolation of pathogenic mycobacterial species from patient samples.…”

“…Genomic data from a number of strains from Ghana has facilitated the development of methods based on the detection of single nucleotide polymorphisms (SNP typing) for studying M. ulcerans transmission pathways and phylogenetic relationships [10] , [11] . However, these methods have only been used successfully with pure bacterial cultures [10] , [11] .…”