Blending DNA binding dyes to improve detection in real-time PCR
نویسندگان
چکیده
The success of real-time PCR (qPCR) analysis is partly limited by the presence of inhibitory compounds in the nucleic acid samples. For example, humic acid (HA) from soil and aqueous sediment interferes with amplification and also quenches the fluorescence of double-stranded (ds) DNA binding dyes, thus hindering amplicon detection. We aimed to counteract the HA fluorescence quenching effect by blending complementary dsDNA binding dyes, thereby elevating the dye saturation levels and increasing the fluorescence signals. A blend of the four dyes EvaGreen, ResoLight, SYBR Green and SYTO9 gave significantly higher fluorescence intensities in the presence and absence of HA, compared with the dyes applied separately and two-dye blends. We propose blending of dyes as a generally applicable means for elevating qPCR fluorescence signals and thus enabling detection in the presence of quenching substances.
منابع مشابه
Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature
The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis. Al...
متن کاملApplication of Rapid and Sensitive Real Time PCR Technique in Detection of DNA Impurities in Recombinant Interferon
Background & Objective: Interferon belongs to a family of cytokines, which has the most important role in the innate immune response to virus infections. While producing recombinant interferon in biological host, some pieces of host nucleic acids remain in product. Because of limitations in previous techniques for detection of these impurities, the objective of this study is to use rapid ...
متن کاملEvaluation of Microscopy Sensitivity, Specificity in Detection of P. falciparum and P. vivax, Using Monoplex real-time PCR, Gezira, Sudan
Background: Malaria is still account for 200 million cases annually. Microscopy is the gold standard technique for malaria parasites detection. PCR-based techniques can detect malaria infections with high sensitivity. The study aimed to evaluate the sensitivity of microscopy technique in the detection of P. falciparum and P. vivax, using monoplex real-time PCR, Gezira State, Central Sudan. Met...
متن کاملQuantification and Optimization of Candida albicans DNA in Blood Samples Using Real- Time PCR
Background: Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis. Methods: Five milliliter blood samples from...
متن کاملEprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis
Real-time monitoring of PCR is one of the most important methods for DNA and RNA detection widely used in research and medical diagnostics. Here we describe a new approach for combined real-time PCR monitoring and melting curve analysis using a 3' end-blocked Exciton-Controlled Hybridization-sensitive fluorescent Oligonucleotide (ECHO) called Eprobe. Eprobes contain two dye moieties attached to...
متن کامل