Molecular inversion probes for sensitive detection of Mycobacterium tuberculosis.
Nucleic acid-based detection of Mycobacterium tuberculosis infections has the potential to improve the analysis of the tuberculosis epidemiology and patient care by increasing the specificity and sensitivity of diagnosis. One potential diagnostic sequence, the DR locus, is present in all isolates of M. tuberculosis complex bacteria. It encodes no known gene product but is useful for molecular typing of M. tuberculosis because of its fortuitous absence in non-tuberculosis strains of mycobacteria. The DR locus contains a variable number of short direct repeats interspersed with non-repetitive spacers and is commonly used as a target for the spoligotyping method, a technique based on the detection of the presence or absence of distinct spacers between the repeats. In this study, we attempted to combine the specificity of molecular inversion probe (MIP) technology with the sensitivity of modified pyrosequencing readout in order to detect a short conserved 18 bp sequence included in DR locus in 25 isolates of M. tuberculosis. Additional sensitivity was obtained by introducing modifications in pyrosequencing methodology, by these means we achieved to detect 500 fg of M. tuberculosis DNA.
Novais
Rogerio C
RC
Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. rnovais@uerj.br
Borsuk
Sibelle
S
Dellagostin
Odir A
OA
Thorstenson
Yvonne R
YR
D015373:Bacterial Typing Techniques; D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D011756:Diphosphates / Q000378:metabolism; D006801:Humans; D007287:Inorganic Chemicals / Q000378:metabolism; D015336:Molecular Probe Techniques; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009841:Oligonucleotides / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology
D004269:DNA, Bacterial; D011756:Diphosphates; D007287:Inorganic Chemicals; D015335:Molecular Probes; D009841:Oligonucleotides