Lineage specific FMDV Real-time RT-PCR assays

November 2017

This set of real-time RT-PCR type-specific as-says was designed for the detection of FMDV strains currently circulating in West Eurasia (Reid et al., 2014). The set of real-time RT-PCR assays aims to detect FMDV O/ME-SA/PanAsia-2, A/ASIA/Iran05, Asia1/ASIA/Group 1, 2 and 6. 

Designed to detect: O/ME-SA/PanAsia-2, A/ASIA/Iran- 05, Asia1/ASIA/Group 1, Asia1/ASIA/Group 2 and Asia1/ASIA/Group 6

Primers and probes are listed in the table below.

* Probe oligonucleotide should be ordered to contain the appropriate reporter and quencher for your real time PCR system.

Thermal Profile:

Amplification of reactions is to be carried out using a real-time PCR instrument under following conditions:

• 60°C for 30 min,
• 95°C for 10 min
• followed by 50 cycles of 95°C for 15 sec and 60°C for 1 min.

Fluorescence data is collected at the annealing/elongation step.

November 2017

This FMDV-O Ind-2001-specific real-time RT-PCR is a molecular tool for detection of foot-and-mouth Disease virus O/ME-SA/Ind2001 lineage (Knowles et al., 2014). This lineage was originally detected on the Indian sub-continent, but has since spread westwards through the Middle East to North Africa and eastwards into Southeast Asia.

Designed to detect: O/ME-SA/Ind-2001d

Primers and probes are listed in the table below.

* Probe oligonucleotide should be ordered to contain the appropriate reporter and quencher for your real time PCR system.

Thermal Profile:

Amplification of reactions is to be carried out using a real-time PCR instrument under following conditions:

• 60°C for 30 min,
• 95°C for 10 min
• followed by 50 cycles of 95°C for 15 sec and 60°C for 1 min.

Fluorescence data is collected at the annealing/elongation step.

January 2025

The following real-time RT-PCR assay is designed to specifically detect the O/ME-SA/SA-2018 lineage of foot-and-mouth disease virus (FMDV).

Designed to detect: O/ME-SA/SA-2018

* Probe oligonucleotide should be ordered to contain the appropriate reporter and quencher for your real time PCR system.

November 2017

This FMDV-A lineage-VII-specific real-time RT-PCR is a molecular tool for detection of foot-and-mouth Disease virus A/ASIA/G-VII lineage (Saduakassova et al., 2018). This lineage was originally detected on the Indian sub-continent, but has since spread through western Asia.

Designed to detect: A/ASIA/G-VII

Primers and probes are listed in the table below.

* Probe oligonucleotide should be ordered to contain the appropriate reporter and quencher for your real time PCR system.

Thermal Profile:

Amplification of reactions is to be carried out using a real-time PCR instrument under following conditions:

• 60°C for 30 min,
• 95°C for 10 min
• followed by 50 cycles of 95°C for 15 sec and 60°C for 1 min.

Fluorescence data is collected at the annealing/elongation step.

March 2025

In response to the emergence of SAT 1/I in the Middle East during 2025, the following real-time RT-PCR assay (as described by Bachanek-Bankowska et al., 2016) has been tested for representative samples from the region. Data from WRLFMD indicate that this assay is able to detect viruses from the SAT 1/I lineage.

Designed to detect: SAT 1/I

* Probe oligonucleotide should be ordered to contain the appropriate reporter and quencher for your real time PCR system.

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Assay protocol and amplification conditions:

This SAT1 assay was designed to have identical reagent and cycling conditions as the pan-FMDV real-time RT-PCR assay described in the WOAH manual (Callahan et al., 2002) and testing to date at the WRLFMD indicates that the assay produces Ct values broadly equivalent to the Callahan 3D pan-FMDV assay. Therefore, to perform this test, we suggest that laboratories use their existing real-time RT-PCR strategies (reagents and cycling conditions) and simply replace the 3D or 5’UTR primers and probe with the primers and probe described above. This should be done in conjunction with a pan-FMDV real-time RT-PCR assay.

SAT 1/III: June 2026

https://sitesv2.anses.fr/en/minisite/lrue-fievre-aphteuse/new-rt-rt-pcr-detection-sat1iii-fmdv-candidate-assay
https://sitesv2.anses.fr/en/minisite/lrue-fievre-aphteuse/rtrt-pcr-detection-sat1iii-%E2%80%9Cbot177-%E2%80%9D

Designed to detect: SAT 1/III

The SAT1/III primers and probe designed by ANSES , the European Union Reference Laboratory for FMD, were evaluated at the WRLFMD, Pirbright, UK. The assay successfully detected SAT1/III viruses collected in Lebanon (2025) and Azerbaijan (2025).

No signal was observed when testing viruses of the following lineages, geographical origins and years of collection:

April 2026

https://sitesv2.anses.fr/en/minisite/lrue-fievre-aphteuse/new-rt-rt-pcr-detection-sat1iii-fmdv-candidate-assay
https://sitesv2.anses.fr/en/minisite/lrue-fievre-aphteuse/rtrt-pcr-detection-sat1iii-%E2%80%9Cbot177-%E2%80%9D

Designed to detect: SAT 1/III

This real-time RTP-PCR is a molecular tool for the detection of foot-and-mouth disease virus SAT 1/III topotype and was developed at ANSES, the European Union Reference Laboratory for FMD.

Further testing/evaluation of this assay is underway and as more data becomes will this will be updated.

Designated to detect: SAT 1/III

November 2017

This real-time RT-PCR is a molecular tool for detection of Foot-and-mouth disease virus SAT 2/VII topotype and was developed in response to the SAT 2 outbreak in Egypt in 2012 (Ahmed et al., 2012). Outside of Egypt this lineage has been detected in Libya, Palestinian Autonomous Territories, Eritrea and Cameroon.

Designed to detect: SAT 2/VII

Primers and probes are listed in the table below.

* Probe oligonucleotide should be ordered to contain the appropriate reporter and quencher for your real time PCR system.

Thermal Profile:

Amplification of reactions is to be carried out using a real-time PCR instrument under following conditions:

• 60°C for 30 min,
• 95°C for 10 min
• followed by 50 cycles of 95°C for 15 sec and 60°C for 1 min.

Fluorescence data is collected at the annealing/elongation step.

September 2025

https://eurl-fmd.anses.fr/en/minisite/lrue-fievre-aphteuse/rtrt-pcr-detection-sat2v-fmdv

Designed to detect: SAT 2/V

This real-time RTP-PCR is a molecular tool for the detection of foot-and-mouth disease virus SAT 2/V topotype and was developed at ANSES, the European Union Reference Laboratory  for FMD.

Designated to detect: SAT 2/V

February-March 2023

FMDV SAT 2 outbreaks in Iraq and Jordan due to SAT2/XIV. The results presented here are a preliminary evaluation of a second real-time RT-PCR assay for the detection of SAT2/XIV viruses that have caused these outbreaks

Designed to detect: SAT 2/XIV

Primers and probes are listed in the table below.

* Probe oligonucleotide should be ordered to contain the appropriate reporter and quencher for your real time PCR system.

7 March 2023

Results from initial testing of new assay at the WRLFMD

This latest assay has been shown to be able to detect nucleic acid extracted from a sample containing SAT/XIV FMD virus from Iraq where Ct values were broadly equivalent to the results from the pan-serotypic 3D real-time RT-PCR. Preliminary lineage specificity testing indicates that this assay does not detect FMD viruses from serotype O (O/EA-3, O/ME-SA/PanAsia-2^ANT-10, O/ME-SA/PanAsia-2^QOM-15, O/ME-SA/Ind-2001e), serotype A (A/ASIA/Iran-05),  serotype Asia 1 (Sindh-08) and serotype SAT2 (VII).