Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.
Judith Kamerbeek,Leo M. Schouls,Arend H. J. Kolk,M van Agterveld,D. van Soolingen,Sjoukje Kuijper,Annelies Bunschoten,H Molhuizen,R J Shaw,M Goyal,J. D. A. Van Embden +10 more
TLDR
A novel method based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.Abstract:
Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.read more
Citations
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CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats.
TL;DR: CRISPRFinder is described, a web service offering tools to detect CRISPRs including the shortest ones including one or two motifs, define DRs and extract spacers, and get the flanking sequences to determine the leader.
Journal ArticleDOI
Identification of genes that are associated with DNA repeats in prokaryotes.
TL;DR: A novel family of repetitive DNA sequences that is present among both domains of the prokaryotes but absent from eukaryotes or viruses is studied, characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non‐repetitive sequences.
Journal ArticleDOI
Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.
TL;DR: The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA.
Journal ArticleDOI
Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis
Philip Supply,Philip Supply,Caroline Allix,Sarah Lesjean,Sarah Lesjean,Mara Cardoso-Oelemann,Mara Cardoso-Oelemann,Sabine Rüsch-Gerdes,Eve Willery,Eve Willery,Evgueni Savine,Evgueni Savine,Petra E. W. de Haas,Henk van Deutekom,Solvig Roring,Pablo Bifani,Natalia Kurepina,Barry N. Kreiswirth,Christophe Sola,Nalin Rastogi,Vincent Vatin,Vincent Vatin,Maria Cristina Gutierrez,Maryse Fauville,Stefan Niemann,Robin A. Skuce,Kristin Kremer,Camille Locht,Camille Locht,Dick van Soolingen +29 more
TL;DR: A discriminatory subset of 15 loci with the highest evolutionary rates was defined that concentrated 96% of the total resolution obtained with the full 24-locus set, and its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing.
Journal ArticleDOI
CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.
TL;DR: The authors have sequenced a total of 109 alleles of the three Y. pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate, and it appears that addition of new motifs to a common ancestral element is the most frequent event.
References
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