The TaqMan® MGB probe for Streptococcus mutans was designed from sequence analyses, and the primers were the same as nested PCR. The first round of nested PCR was carried out with the bacterial universal primers, while a second PCR was conducted by using primers specific for the 16S rRNA gene of Streptococcus mutans. Conventional nested PCR and TaqMan® MGB real-time PCR were applied independently. We extracted six DNA samples from different streptococcal strains for PCR reaction. To design a new TaqMan® MGB probe for improving the specificity of Streptococcus mutans's detection. Zheng, Hui Lin, Jiu-Xiang DU, Ning Chen, Feng We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates. The utility of the dual- probe approach was demonstrated on practical examples by using field specimens. On the other hand, the second probe additively contributed to the overall fluorescence signal. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single- probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. Specific concerns are related to false negativity due to probe binding failure. Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Nagy, Alexander Vitásková, EliÅ¡ka ÄŒernÃková, Lenka KÅ™ivda, Vlastimil JiÅ™incová, Helena Sedlák, Kamil HornÃÄková, Jitka HavlÃÄková, Martina In conclusion, a simple, rapid method, with high specificity and stability, for the detection of the KIT genotype in pigs was established using TaqMan MGB probe real-time quantitative PCR.Įvaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay Furthermore, use of the MGB probe set resulted in detection of the target mutation at a high resolution and stability standard curves illustrated that the amplification efficiencies of KIT1 (G) and KIT2 (A) were approximately equal (98.8% and 97.2%). The amplification efficiencies for KIT and ESR were approximately equal, indicating ESR was an appropriate control gene. The CT values among dilutions were significantly different (p < 0.001) and the coefficients of variation from each dilution were low (from 0.13% to 0.26%). A series of parallel amplification curves with the same internal distances were obtained using gradually diluted DNA as templates. Based on the sequence of the resulting amplified fragment, an MGB probe set was designed to detect the ratio of splice mutation to normal using FQ-PCR. Second, to detect the splice mutation ratio of the G:A substitution in intron 17, a 175 bp region, including the target mutation, was amplified from genomic DNA. A real-time fluorescence-based quantitative PCR (FQ-PCR) protocol was developed to accurately detect KIT CNVs. The single-copy gene, estrogen receptor (ESR), was used as an internal control. First, to detect KIT copy number variation (CNV), primers for exon 2 of the KIT gene, along with a TaqMan minor groove binder ( MGB) probe, were designed. The purpose of this study was to establish a simple, rapid method to determine KIT genotype in pigs. The dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene: a 450 kb duplication containing the entire KIT gene together with flanking sequences and one splice mutation with a G:A substitution in intron 17. Li, Xiuxiu Li, Xiaoning Luo, Rongrong Wang, Wenwen Wang, Tao Tang, Hui Detection of KIT Genotype in Pigs by TaqMan MGB Real-Time Quantitative Polymerase Chain Reaction.
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