A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1-ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was compl...

The recent development of real-time PCR has offered the opportunity of sensitive and accurate quantification of mRNA levels that is crucial in biomedical research. Although reverse transcription (RT)-PCR is at present the most sensitive method available, many low abundant mRNAs are, although detectable, often not quantifiable. Here we report an improved two-step real-time RT-PCR procedure using...

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...

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...

Infectious bursal disease (IBD) is an immunosuppressive, acute and highly contagious illness of growing-poultry stock infected with infectious bursal disease virus (IBDV). It is common in Pakistan, causing potential economic losses throughout the year. The objective of the study is to propose a rapid, sensitive and specific diagnostic tool, and compare it with existing commonly used reverse tra...

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...

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...

We developed a one-step, single-tube genogroup-specific reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay for the detection of group B Coxsackie genomes targeting 5’ UTR region. The amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification...

Summary To overcome the detection limits inherent to DNA array-based methods of transcriptome analysis, we developed a real-time reverse transcription (RT)-PCR-based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene-specific primer pairs and SYBR Green to monitor amplification of double-stranded DNA (dsDNA), transcri...

EvaGreen is a new DNA intercalating dye successfully used in quantitative real-time PCR. In the present work, we firstly apply EvaGreen to the analysis of dsDNA by CE with LIF detection. Comparisons of EvaGreen dye with the commonly used dyes SYBR Green I and SYBR Gold were preformed in dsDNA analysis by CE. The linear range of dsDNA using EvaGreen was slightly wider than that using SYBR Gold a...