Human papillomavirus status was determined by a nested real-time PCR assay using consensus HPV primers PGMY09/11/HMB01 (Gravitt et al, 2000) and GP5+/6+ primers (de Roda Husman et al, 1995).
To determine if the DNA extracts exhibited low level non-specific inhibition of PCR, 10 samples were subjected to 30 cycles of the first round hBG PCR (reaction mix and conditions as above) followed by 40 cycles of a nested real-time SYBR-green PCR using the SYBR-green Fast PCR kit (Roche, Lewes UK) according to the manufacturer's instructions.
We report here the development of a nested real-time PCR for the detection and genotyping of WNV strains by means of dissociation-curve analysis, using fluorescence resonance energy transfer (FRET) probe technology.
A nested real-time reverse transcription PCR (RT-PCR) specific for the WNV NS5 gene that distinguishes between lineages 1 and 2 with WNV-specific FRET probes on the basis of dissociation curve analysis was used to screen specimens (23 ).
This is also true of a two-step nested real-time PCR developed by the group of Jalal in 2007 which targeted the omp-1 gene and used eleven probes for genotyping the serovars D-K and L1, L2 and L3 in the second, nested step [ 34].
A new highly sensitive nucleic acid amplification test, combined nested and real-time polymerase chain reaction (PCR) in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantage of two PCR techniques, i.e., nested PCR and real-time PCR.
A 10-µL aliquot of the DNA extract was used in real-time PCR, and results were confirmed by using a nested PCR and sequencing as described (9 – 11 ).
The performance was evaluated using a nested 10-fold cross-validation repeated 20 times.
Env was amplified using a nested PCR.
The amplicon (0.2µl) from the first PCR product was used as template for a nested quantitative real-time PCR (45 cycles at 95°C for 5 s, 54°C for 10 s, and 72°C for 15 s; acquisition was at 83°C) conducted using the LightCycler-RNA amplification kit SYBR Green 1 (Roche, IN).
