Dataset Information

Detection of Mycobacterium ulcerans by real-time PCR with improved primers.

ABSTRACT: BACKGROUND:Buruli ulcer is a severe skin disease caused by Mycobacterium ulcerans. Real-time PCR targeting the IS2404 sequence has been used as a reliable and rapid method for the diagnosis of Buruli ulcer and detection of M. ulcerans in the environment. The genome of M. ulcerans contains hundreds of IS2404 copies, which have variability in certain sequences. Therefore, the design of new primers specific to conserved IS2404 regions may potentially improve the sensitivity of M. ulcerans detection and, consequently, the diagnosis of Buruli ulcer, thus ensuring timely treatment of the disease. RESULTS:In silico analysis indicates that DNA sequences of the IS2404 elements are highly variable within a single strain. As the binding sites of conventional IS2404-specific primers used for M. ulcerans detection contain polymorphic sequences, we designed new primers, which enabled the detection of M. ulcerans by real-time PCR with higher sensitivity and similar specificity with respect to that of conventional primers. However, the increase in sensitivity with the new primers depended on the M. ulcerans strain. CONCLUSIONS:The results suggest that real-time PCR based on the new primers could improve Buruli ulcer diagnosis and M. ulcerans detection in environmental samples.

SUBMITTER: Shinoda N

PROVIDER: S-EPMC5009631 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Detection of Mycobacterium ulcerans by real-time PCR with improved primers.

Shinoda Noriko N Nakamura Hajime H Watanabe Mineo M

Tropical medicine and health 20160819 1

<h4>Background</h4>Buruli ulcer is a severe skin disease caused by Mycobacterium ulcerans. Real-time PCR targeting the IS2404 sequence has been used as a reliable and rapid method for the diagnosis of Buruli ulcer and detection of M. ulcerans in the environment. The genome of M. ulcerans contains hundreds of IS2404 copies, which have variability in certain sequences. Therefore, the design of new primers specific to conserved IS2404 regions may potentially improve the sensitivity of M. ulcerans d ...[more]

PMID: 27610043

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Project description:BACKGROUND: Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora. RESULTS: Design of specific primers and probe was achieved based on comparison of 108 published bifidobacterial 16S rDNA sequences with the recently published sequence of the human faecal isolate B. thermophilum RBL67. Specificity of the primer was tested in silico by similarity search against the sequence database and confirmed experimentally by PCR amplification on 17 Bifidobacterium strains, representing 12 different species, and two Lactobacillus strains. The qPCR assay developed was linear for B. thermophilum RBL67 DNA quantities ranging from 0.02 ng/microl to 200 ng/microl and showed a detection limit of 10(5) cells per gram faeces. The application of this new qPCR assay allowed to detect the presence of B. thermophilum in one sample from a 6-month old breast-fed baby among 17 human faecal samples tested. Additionally, the specific qPCR primers in combination with selective plating experiments led to the isolation of F9K9, a faecal isolate from a 4-month old breast-fed baby. The 16S rDNA sequence of this isolate is 99.93% similar to that of B. thermophilum RBL67 and confirmed the applicability of the new qPCR assay in faecal samples. CONCLUSION: A new B. thermophilum-specific qPCR assay was developed based on species-specific target nucleotides in the 16S rDNA. It can be used to further characterize the composition of the bifidobacterial community in the human gastrointestinal tract. Until recently, B. thermophilum was considered as a species of animal origin, but here we confirm with the application of this new PCR assay the presence of B. thermophilum strains in the human gut.

Dataset Information

Detection of Mycobacterium ulcerans by real-time PCR with improved primers.

Publications

Detection of Mycobacterium ulcerans by real-time PCR with improved primers.

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