Use of Sloppy Molecular Beacon Probes for Identification of Mycobacterial Species

Abstract

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 (T m ) 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 T m 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.A classic approach for determining the identity of a bacterial species is to employ PCR to exponentially amplify a selected segment of a 16S rRNA gene (26), utilizing a pair of "universal primers" that bind to highly conserved sequences at the ends of the target region, and to then use a sophisticated technique to identify a species-specific sequence in the middle of the resulting amplicons (12). These identification methods include nucleotide sequence analysis (27) and electrophoretic determination of the sizes of fragments produced by incubation of the amplicons with selected restriction endonucleases (17). However, these techniques are time consuming, costly, and labor intensive.A more desirable approach is to use a set of fluorescently labeled, sequence-specific oligonucleotide hybridization probes, each of which hybridizes to a different species-specific sequence under conditions in which only perfectly complementary probe-target hybrids form.In a screening assay, only one probe in the set will form a hybrid, thereby identifying the species that is present. This method is rapid and can be carried out in sealed reaction tubes, utilizing probes possessing interacting label moieties, such as TaqMan probes (10), LightCycler probes (24), or molecular beacons (19), all of which generate distinctive fluorescence signals when they hybridize to complementary target amplicons. Multiplex PCR screening assays that contain a number of different probes can also be prepared, each probe specific for a different species and each possessing a differently colored fluorophore (21, 22). However, a comprehensive screening procedure requires upwards of 100 different species-specific probes and has to be carried out in many PCR assay tubes. Although arrays of probes on hybridization chips have been utilized in screening assays to determine the identity of 16S rRNA amplicons (5), this approach is nonhomogeneous, thereby risking the cross contamination of untested samples, and has not been adopted by clinical laboratories.A particularly attrac...

Simplified Microarray System for Simultaneously Detecting Rifampin, Isoniazid, Ethambutol, and Streptomycin Resistance Markers in Mycobacterium tuberculosis

“…At least one near-neighbor probe was sensitive to three of the six known mutations that were not represented by a specific probe on the microarray, which is similar to the behavior of "sloppy molecular beacon" probes (22). There were also 96 wild-type sequences interrogated by the microarray test, 100% of which were correctly identified as wild type.…”

“…It is usually assumed that hybridization specificity needs to be perfect in order for microarrays to have diagnostic value. On the other hand, "sloppy molecular beacons" that are responsive to multiple mutations within a 20-mer reporting probe (22) form the basis of the GeneXpert MTB/RIF assay and enable reasonably sensitive detection (but not identification) of mutations regardless of where the mutation occurs within the reporting probe.…”

Rapid Detection of FKS -Associated Echinocandin Resistance in Candida glabrata

“…Multiple PCR assays will therefore be needed, and optimization of assay conditions is extremely demanding due to complex oligonucleotide interactions in a multiplex format. In contrast, a more desirable and feasible approach to distinguish between closely related genotypes is to use asymmetric PCR in conjunction with MB probe-based melting curve analysis (14,15). During asymmetric PCR, a singlestranded amplicon is produced, allowing the probe to anneal and generate fluorescence at low temperature.…”

Demonstrating a Multi-drug Resistant <em>Mycobacterium tuberculosis</em> Amplification Microarray

“…We suspect that this type of probe behavior is a consequence of secondary and tertiary structure in the single-stranded amplicon and/or probe 34 , and is not something that can be predicted a priori. Nevertheless, probe sensitivity to near neighbor mutations is analogous to the use of sloppy molecular beacons to detect multiple drug resistance mutations with a minimal set of real-time PCR probes 35,36 , and can be diagnostically advantageous provided the test does not generate false positives relative to the phenotypic drug susceptibility. …”