ASFV DNA Quantification Protocol

DNA was extracted, using a MagMAX extraction system (Thermo Scientific) and a MagVet universal isolation kit (Life Technologies), from whole peripheral blood that had been collected in EDTA-containing tubes at different days postimmunization. qPCR was carried out on a Stratagene Mx3005P system (Agilent Technologies, Santa Clara, CA, USA) following a protocol modified from that of King et al. (39 (link), 51 (link)) using the primers Vp72 sense (CTG CTC ATG GTA TCA ATC TTA TCG A) and Vp72 antisense [GAT ACC ACA AGA TC(AG) GCC GT] and the probe 5′-(6-carboxyfluorescein [FAM])-CCA CGG GAG GAA TAC CAA CCC AGT G-3′-(6-carboxytetramethylrhodamine [TAMRA]).

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Reis A.L., Goatley L.C., Jabbar T., Sanchez-Cordon P.J., Netherton C.L., Chapman D.A, & Dixon L.K. (2017). Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge. Journal of Virology, 91(24), e01428-17.

Publication 2017

MagVet universal isolation kit Stratagene Mx3005P system

6 carboxyfluorescein 6 carboxytetramethylrhodamine Blood Edta Isolation Primers

Corresponding Organization :

Other organizations : The Pirbright Institute

Top 5 similar protocols

Variable analysis

independent variables

Days postimmunization

dependent variables

Quantitative PCR (qPCR) results

control variables

Whole peripheral blood collected in EDTA-containing tubes
DNA extraction using MagMAX extraction system and MagVet universal isolation kit
QPCR performed on Stratagene Mx3005P system
Primers Vp72 sense and Vp72 antisense
Probe 5′-(6-carboxyfluorescein [FAM])-CCA CGG GAG GAA TAC CAA CCC AGT G-3′-(6-carboxytetramethylrhodamine [TAMRA])

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