A novel endonuclease IV post-PCR genotyping system

Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5′ end and fluorophore attached to the 3′ end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3′ end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection

Sensitive and specific assay for the simultaneous detection of Mycoplasma genitalium and macrolide resistance-associated mutations

Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg MacrolideR qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other micro-organisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg MacrolideR qPCR. The Mg MacrolideR qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg MacrolideR qPCR, indicating specificity. The Mg MacrolideR qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics

Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse™ Probe System in challenging sequence environment

Abstract Probe and primer design for single nucleotide polymorphism (SNP) detection can be very challenging for A-T DNA-rich targets, requiring long sequences with lower specificity and stability, while G-C-rich DNA targets present limited design options to lower GC-content sequences only. We have developed the MGB Eclipsee™ Probe System, which is composed of the following elements: MGB Eclipse probes and primers, specially developed software for the design of probes and primers, a unique set of modified bases and a Microsoft Excel macro for automated genotyping, which ably solves, in large part, this challenge. Fluorogenic MGB Eclipse probes are modified oligo-nucleotides containing covalently attached duplex-stabilising dihydrocyclopyrroloindole tripeptide (DPI3), the MGB ligand (MGB™ is a trademark of Epoch Biosciences, Bothell, WA), which has the combined properties of allowing the use of short sequences and providing great mismatch discrimination. The MGB moiety prevents probe degradation during polymerase chain reaction (PCR), allowing the researcher to use real time data; alternatively, hybridisation can be accurately measured by a post-PCR two-colour melt curve analysis. Using MGB Eclipse probes and primers containing modified bases further enhances the analysis of difficult SNP targets. G- or C-rich sequences can be refractory to analysis due to Hoogsteen base pairing. Substitution of normal G with Epoch's modified G prevents Hoogsteen base pairing, allowing both superior PCR and probe-based analysis of GC-rich targets. The use of modified A and T bases allows better stabilisation by significantly increasing the Tm of the oligonucleotides. Modified A creates A-T base pairs that have a stability slightly lower than a G-C base pair, and modified T creates T-A base pairs that have a stability about 30 per cent higher than the unmodified base pair. Together, the modified bases permit the use of short probes, providing good mismatch discrimination and primers that allow PCR of refractory targets. The combination of MGB Eclipse probes and primers enriched with the MGB ligand and modified bases has allowed the analysis of refractory SNPs, where other methods have failed.</p