rtRT-PCR for the detection of O/ME-SA/SA-2018 FMDV

This real-time RT-PCR is a molecular tool for detection of Foot-and-mouth disease virus lineage O/ME-SA/SA-2018, as it is an emerging lineage in South Asia since 2018. The assay has been tested on 34 FMDV positive samples (including 12 SA-2018 samples) with a specificity of 91,7% (11/12 SA-2018 samples detected). The primers and probes are indicated hereafter: 

Oligo name

(final concentration)

Sequence (5’-3’)

Use

SA2018_F3 (0.4 μM)

ACAACACCACCAATCCAAC

Forward Primer

SA2018_P3 (0.3 μM)

FAM-ACTCACCCGACTTGCACTGCCGT-TAMRA

Probe 

SA2018_Rev2 (0.4 μM)

CGTTGTAAACAGTAGCCATGA

Reverse Primer

The SA-2018 has been validated using Ag-Path kit in a duplex system with β-actin, and following the volumes and concentrations as follow (5 µl of RNA): 

 

Volume (µl) 

Concentration 

 

For one tube 

Initial 

Final 

Ultrapure water (DNase RNase Free) 

1,15 

/ 

Buffer 2X (kit AgPath-ID™) 

12,5 

2 

1 

X 

Primer F 

1 

10 

0,4 

µM 

Primer R 

1 

10 

0,4 

µM 

Probe FAM-TAMRA 

0,75 

10 

0,3 

µM 

Primer F β-actine 

1 

10 

0,4 

µM 

Primer R β-actine 

1 

10 

0,4 

µM 

Probe VIC-TAMRA β-actine 

0,6 

5 

0,12 

µM 

RT-PCR mix 25X (Enzyme) 

1 

25 

1 

X 

Real-time PCR program: 

Cycles of RTq-PCR 

T° 

Time 

nb cycles 

45°C 

10min 

1 

95°C 

10min 

1 

95°C 

15s 

45 

62°C 

1min 

 

 NB: This system has been validated on a small number of samples and should therefore be tested against other samples from this lineage.