Classification of fowl adenoviruses by use of phylogenetic analysis and high-resolution melting-curve analysis
of the hexon L1 gene region.
was performed using 95[degrees]C for 60 s, 40[degrees]C for 20s, and heating to 80[degrees]C with a ramp rate of 0.03[degrees]C/s with continuous fluorescence acquisition.
by Using Hybridization Probes
The [T.sub.c] for fast COLD-PCR for a given amplicon is defined by first amplifying a wild-type sample via conventional PCR and then conducting a melting-curve analysis
(ramping at 0.2 [degrees]C/s from 65 [degrees]C-98 [degrees]C) to identify the [T.sub.m].
The presence of a KRAS mutation is detected either by an increase of fluorescence during PCR amplification or by post-PCR fluorescent melting-curve analysis
The amplification program was followed by a melting-curve analysis
consisting of 15-s holds at 95 [degrees]C, 45 [degrees]C, and 95 [degrees]C.
High-resolution melting-curve analysis
(HRM) is a simple and cost-effective post-PCR technique that can be used for methylation profiling (20).
The assay ended with a melting-curve analysis
: heating to 95[degrees]C without a hold, rapid ramping to 59[degrees]C and holding for 30 s, gradual heating to 95[degrees]C at 0.1[degrees]C/s, and final cooling to 40[degrees]C.
was performed by use of previously described methods (7) with LightScanner Software (version 188.8.131.524).
Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis
. Arch Virol 2005;150: 2429-38.
We tested primer pairs by analyzing the amplification products by standard agarose gel electrophoresis and by melting-curve analysis
, as described.
In contrast to traditional melting-curve analysis
, high-resolution melting with the HR-1 instrument reliably detects single-base differences in homozygous and heterozygous sequences (17).