A screening system including a new real-time PCR assay for the monitoring of influenza A virus in wild birds was developed. The real-time PCR assay uses SYBR green chemistry and the primers are targeting the matrix gene of influenza A virus. The performance of the assay was compared with two other assays, one assay also using SYBR green chemistry and one assay using TaqMan chemistry, i.e. a spe...

A real-time multiplex PCR procedure with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid and reliable identification of Plum pox virus (PPV) isolates of strains D and M. Members of the different strains were identified by their distinctive melting temperatures (T(m)s); 84.3-84.43 degrees C for D isolates, and 85.34-86.11 degrees C for M isolates. T...

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons du...

Real-time RT-PCR and SYBR green I melt curve analysis of a 74 bp amplicon enabled identification of Plum pox virus strains C, EA, and W, with distinct T(m)'s associated with each strain. This test is a useful supplement to a real-time RT-PCR test described earlier that was used to distinguish PPV strains D and M. A longer fragment of 155 bp was not effective for strain identification. A simplif...

Doxycycline, a new generation tetracycline antibiotic, is currently one of choice for the treatment and prevention of infections caused by agents of biowarfare. We are developing real time PCR assays to detect tetracycline resistance genes in Gram-negative bacteria. The assay was developed as a multiplex SYBR Green I detection using the Roche Lightcycler and multi-melting peak analysis followed...

Flow cytometry can rapidly quantitate bacteria and discriminate them from debris. Researchers at the Swiss Federal Institute of Aquatic Science and Technology (Eawag, Switzerland) have developed a standard flow cytometric staining protocol and a corresponding BD AccuriTM C6 software analysis template to discriminate bacteria from debris in drinking water samples. The template employs a single, ...

the sscp technique is based on the appearance of new “refolding” conformations during electro-phoresis due to mutation. in order to develop a simple, non-radioactive sscp analysis method so that it can reliably detect single nucleotide changes in pcr products up to 500 bp in length, extensive optimisation trials were performed . the best separation of sscp bands of pcr products up to 500 bp in ...

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between t...

The accuracy and repeatability of freezing rates and effects of evaporation were examined using a new cryomicroscope system to establish its usefulness in assessing the development of cryopreservation protocols for bovine semen. Post-thaw sperm plasma membrane integrity, as assessed by using combinations of fluorescent stains and flow cytometry, was used in evaluating protocols for freezing spe...