Use of Sloppy Molecular Beacon Probes for Identification of Mycobacterial Species

We report here the use of novel "sloppy" molecular beacon probes in homogeneous PCR screening assays in which thermal denaturation of the resulting probe-amplicon hybrids provides a characteristic set of amplicon melting temperature (Tm) values that identify which species is present in a s...

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Bibliographic Details
Published in:	Journal of Clinical Microbiology 2009-04, Vol.47 (4), p.1190-1198
Main Authors:	El-Hajj, Hiyam H, Marras, Salvatore A.E, Tyagi, Sanjay, Shashkina, Elena, Kamboj, Mini, Kiehn, Timothy E, Glickman, Michael S, Kramer, Fred Russell, Alland, David
Format:	Article
Language:	eng
Subjects:	Bacteriological Techniques - methods Bacteriology Biological and medical sciences Fundamental and applied biological sciences. Psychology Humans Mass Screening - methods Microbiology Miscellaneous Molecular Probe Techniques Mycobacteriology and Aerobic Actinomycetes Mycobacterium Mycobacterium - classification Mycobacterium - genetics Mycobacterium - isolation & purification Sensitivity and Specificity Transition Temperature
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Summary:	We report here the use of novel "sloppy" molecular beacon probes in homogeneous PCR screening assays in which thermal denaturation of the resulting probe-amplicon hybrids provides a characteristic set of amplicon melting temperature (Tm) values that identify which species is present in a sample. Sloppy molecular beacons possess relatively long probe sequences, enabling them to form hybrids with amplicons from many different species despite the presence of mismatched base pairs. By using four sloppy molecular beacons, each possessing a different probe sequence and each labeled with a differently colored fluorophore, four different Tm values can be determined simultaneously. We tested this technique with 27 different species of mycobacteria and found that each species generates a unique, highly reproducible signature that is unaffected by the initial bacterial DNA concentration. Utilizing this general paradigm, screening assays can be designed for the identification of a wide range of species.
ISSN:	0095-1137 1098-660X