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Plant diseases are often diagnosed by the method of DNA extraction followed by PCR. DNA extraction from plant tissue can be a recalcitrant and lengthy process, and sometimes ends up with inhibitors that reduce PCR amplification efficiency. Here we present a unique approach, RPA-PCR couple, to exclude the DNA extraction step from the standard plant...
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... incubation, reactions were purified with a GeneJET PCR Purification kit (Thermo Scientific, United States) and results were examined by the end point determination method of gel electrophoresis. The forward RPA primer 'ATP9F-M9' and reverse RPA primer 'Phy_Gen_R20' (Table 1) were found to be the optimum RPA primer pair (data not shown). ...Context 2
... primers and probes were ordered from Integrated DNA Technologies, Inc (Belgium). Primers and probe sequences are provided in Table 1, with their locations in the atp9-nad9 region presented in Figure 1. ...Context 3
... preparation was previously described (Munawar et al., 2019) and added in amounts that would be used for assaying plant samples (1 µl). Table S1 in supportive information. DNA extracts concentrations ranged between 50 pg/ µl and 100 pg/ µl and 1µl of each DNA extract was added in individual reaction tubes. ...Context 4
... the UDG system cannot be applied to both RPA and PCR in the couple as the product of RPA is being utilized by the coupled PCR as initial template. Table S1 provides list of the taxa utilized in laboratory evaluations of specificity and their isolate numbers, sources and origins of recovery ...Similar publications
Background Feline Infectious Peritonitis (FIP) is a fatal, systemic disease caused by a mutant form of Feline Infectious Peritonitis virus (FIPV) and has been reported to occur worldwide in domestic cats and massive wild feline species. Meanwhile a definitive diagnosis of FIPV ante mortem remains challenging. The objective was to develop a qPCR for...
Citations
Single nucleotide polymorphisms (SNPs) in human genes are very significant genetic changes and PCR (polymerase chain reaction) or NGS (next-generation sequencing) are extensively employed in SNP analysis. Thanks to the studies on the progress of new technologies, interest in the isothermal nucleic acid amplification approach has increased. As one of these methods, recombinase polymerase amplification (RPA) represents an attractive option for point-of-care nucleic acid quantification. The target SNPs selected within the scope of the study are mutations identified in the PIK3CA gene region (E542K, E545K), and DNA samples which were evaluated about PIK3CA mutations were isolated from the cancer cells MCF7, BT474, and also SKBr3. The optimization studies for the RPA reaction conditions were carried out for parameters such as assay time, temperature, primer, and also magnesium acetate concentration. According to the results of the reaction optimization studies, in which the RPA products can be obtained in the most efficient way, the assay time was determined as 20 min; the temperature as 40°C; the primer concentration as 10 µM and the MgOAc concentration as 140 mM.
