Mycobacterium tuberculosis complex CRISPR genotyping: improving efficiency, throughput and discriminative power of ‘spoligotyping’ with new spacers and a microbead-based hybridization assay

Abstract

The aims of the present study were to implement a microbead-based 'spoligotyping' technique and to evaluate improvements by the addition of a panel of 25 extra spacers that we expected to provide an increased resolution on principal genetic group 1 (PGG 1) strains. We confirmed the high sensitivity and reproducibility of the classical technique using the 43 spacer panel and we obtained perfect agreement between the membrane-based and the microbead-based techniques. We further demonstrated an increase in the discriminative power of an extended 68 spacer format for differentiation of PGG 1 clinical isolates, in particular for the East AfricanIndian clade. Finally, we define a limited yet highly informative reduced 10 spacer panel set which could offer a more cost-effective option for implementation in resource-limited countries and that could decrease the need for additional VNTR (variable number of tandem repeats) genotyping work in molecular epidemiological studies. We also present an economic analysis comparing membrane-based and microbead-based techniques. INTRODUCTIONThe current global plan for tuberculosis (TB) control suggests that it will be difficult to eradicate the disease by 2050 (Dye & Williams, 2008). This issue is particularly relevant to large nations with high TB infection rates, such as Brazil, Russia, India, China and Mexico, but it is also relevant to resource-poor countries in Asia and Africa, which are facing the combined threat of human immunodeficiency virus and TB (Uplekar & Lonnroth, 2007). TB transmission chains may be interrupted in resource-poor countries by improvements in public health programmes, medical diagnostics facilities and provision of adequate drug treatment (Dye & Williams, 2008). The Millenium Development Goals of the United Nations Organization has called for the detection of 70 % of active cases through the development of rapid diagnostics methods, and the attainment of 80 % cure rates, through means such as expansion of the Directly Observed Treatment of Short Course (DOTS+) programme. However, the expanding threat of multidrug resistant (MDR) TB (resistance to Abbreviations: CAS, Central Asian; CRISPR, clustered regularly interspaced palindromic repeats; DR, direct repeat; EAI, East African-Indian; EDC, N-(3-dimethylaminopropyl)-N '-ethylcarbodiimide; LAM, LatinAmerican and Mediterranean; DVR, direct variable repeat; HGDI, Hunter and Gaston index; IGEPE, infection, genetics, emerging pathogens, evolution; MDR, multidrug resistant; MTC, Mycobacterium tuberculosis complex; PGG, principal genetic group; S : N, signal to noise ratio; SNP, single nucleotide polymorphism; TMAC, tetra-methyl ammonium chloride; VNTR, variable number of tandem repeats.A table and figure of spoligotyping data are available as supplementary material with the online version of this paper. Multiplexing is an efficient way to increase the throughput and efficiency of genotyping results and many recent techniques, such as multiple ligation-mediated assays, are showing promise in this area (Bergval e...

Association between living environment and human oral viral ecology

“…They function by acquiring short sequences from invading viruses and utilizing these sequences to resist subsequent exposures to those viruses through nucleic acid interference (Bhaya et al, 2011;Brouns et al, 2008;Hale et al, 2009;Marraffini and Sontheimer, 2009;Sorek et al, 2008;Young et al, 2012). Because CRISPR loci acquire and accumulate short viral sequences (Pourcel et al, 2005;Marraffini and Sontheimer, 2009), they can be used to track viral exposures Andersson and Banfield, 2008;Zhang et al, 2010;Pride et al, 2012b;Rho et al, 2012). In this study, we examined the viromes and CRISPRs from 21 human subjects, 11 independent and 10 from among 4 separate households, in an attempt to gain a better understanding of the role that shared environment has in viral community membership and viral exposures in the human oral cavity.…”

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

“…Common CRISPR-mediated immunity processes are usually defined into three stages: (i) acquisition of a short DNA segment (protospacer) from an invading virus or plasmid, and its insertion at the leader-proximal end of a CRISPR locus (7,8), (ii) generation of small mature CRISPR RNAs (crRNAs) from a longer transcript of a CRISPR locus (9)(10)(11), and (iii) interference of foreign nucleic acid invaders by a crRNA-containing ribonucleoprotein effector complex (12)(13)(14)(15)(16)(17)(18).…”

“…Whereas Cas9 and Csm/Cmr effector complexes directly recognize and nucleolytically degrade invading genes (12,14,16,17,21), Cascade complex by itself is not a nuclease for degradation of invader DNA (13). In Escherichia coli K-12 (type IE), Cascade recognizes and binds crRNA to complimentary sequence in target DNA, generating an RNA mediated displacement loop (R-loop) of single-stranded (ss) DNA and RNA-DNA hybrid within double-stranded (ds) target DNA (13).…”

Legionella pneumophila Sequence Type 1/Paris Pulsotype Subtyping by Spoligotyping

“…That study demonstrated that RFLP-IS can also be used to discriminate Paris pulsotype isolates with a discriminatory index similar to that achieved with spoligotyping. These two methods are membrane-based methods dependent on manual hybridization steps; nevertheless, as described for Mycobacterium, L. pneumophila spoligotyping could be improved by switching from membranes to microbead-based hybridization assays (7,24). Compared to membrane-based assays, these systems allow better standardization of the assays and high-throughput analyses, which should promote the utility of spoligotyping for routinely performed L. pneumophila ST1/Paris pulsotype subtyping.…”