PriProET two-step real-time PCR

Acc.no: L2B9FW | Published: 2008-11-17 by Unknown user | Author: Mikhayil Hakhverdyan

Keywords: swine vesicular disease virus, SVD, SVDV, PriProET, RT-PCR

This report describes a two-step protocol for PriProET real-time PCR amplification of swine vesicular disease viruses (SVDV).

This SOP describes a two-step protocol for PriProET real-time PCR amplification of swine vesicular disease viruses (SVDV). The assay amplifies the 3D-gene of any of SVDV strain while heterologous virus strains including Coxsackievirus B5 remain negative. The sensitivity of assay is five copies of viral genome equivalents. A key point of the assay is tolerance toward mutations in the probe region. Melting curve analysis directly after PCR, with determination of probe melting point, confirms specific hybridisation of the SVDV strains.

The protocol was optimised using ABI 7700 instrument (Applied Biosystems, USA), but later was adopted for RotorGene (Corbett Research, Australia). Both instruments works well with the system, but RotorGene, like many other modern real-time PCR machines, has friendly user format and simplifies analysis of results.

Required Materials

Instructions

RT-PCR

General aspects:

The amplification is based on the TITANIUM Taq DNA polymerase kit (Clontech) with a total reaction volume of 25 µl.

Controls: PC (positive control), NTC (no template control)

After production of the master mix, 23 µl portions are aliquoted into each reaction tube and 2 µl of template is added.

Master mix preparation:

The volume of the master mix depends on the number of extracted samples plus the number of controls (PC; NTC) plus one-two extra reactions.

Before dividing up the master mix into portions of 23 µl per tube, the master mix must be mixed thoroughly and pulse spanned for 5 sec.

Addition of template cDNA:

2 µl template cDNA is added to each tube with 23 µl master mix and close the reaction tubes.

Spin down reaction mix (optional).

Cycler program:

Preliminary denaturation 95°C 2 min

Denaturation 95°C 15 sec

Annealing 60°C 15 sec

55 cycles Elongation 72°C 15 sec

Denaturation 95°C 15 sec

Probe melting profile

96 cycles of [50°C for 10 sec with auto-increments of 0.5°C] for ABI 7700 ?or 40°C->95°C for RotorGene.

Fluorescence channel is FAM for ABI 7700. Filter set for RotorGene is 470nm/610hp (emission/detection)

Fluorescence data are collected in the annealing phase and during the probe melting profile
Analysis of data

A Ct value of approx. 25 should be detectable for PC and a specific probe melting point of 57,5 -72,5°C (depends on the number of mutations in the probe region). Each mutation in the probe region decreases melting point on 5°C.

No Ct value or melting point should be detectable for NTC.

The analysis of field samples is possible if all controls respond as expected.

A suspected SVDV sample is considered as positive in the real-time PCR, if a Ct value is detected for the sample and/or if the FAM fluorescence increases significantly over the base level.

For confirmation of a positive SVDV specific real-time PCR result the probe melting profile has to display a specific probe melting point of 70°C±2.5°C corresponding to a perfect probe match, while Tm =65±2.5°C and Tm =60±2.5°C corresponding to one or two mutations, respectively.