TY - JOUR
T1 - Development and validation of SYBR Green- and probe-based reverse-transcription real-time PCR assays for detection of the S and M segments of Schmallenberg virus
AU - Azkur, Ahmet Kursat
AU - van der Poel, Wim H.M.
AU - Aksoy, Emel
AU - Hakze-van der Honing, Renate
AU - Yildirim, Murat
AU - Yıldız, Kader
PY - 2020/9/1
Y1 - 2020/9/1
N2 - Schmallenberg virus (SBV), discovered in Germany in 2011, causes congenital malformations in ruminants. Reverse-transcription real-time PCR (RT-rtPCR) assays based on various segments of SBV have been developed for molecular detection. We developed alternative RT-rtPCR assays for SBV detection to avoid earlier reported mutations and hypervariable regions of the S and M segments of the viral genome. For SYBR Green-based detection of the S segment, the R2 value and efficiency of the developed assay were 0.99 and 99%, respectively. For probe-based S segment detection, 2 assays were developed; the first had an R2 value of 0.99 and 102% efficiency, and the second had a R2 value of 0.98 and 86% efficiency. The probe-based M segment assay had an R2 value of 1.00 and 103% efficiency. Detection limits of the RT-rtPCR assays with new primer sets were 102 and 101 copies/µL for the S and M segments, respectively. Field samples from cattle and sheep were also used for primary validation of the developed assays. Our assays should be suitable for SBV detection in ruminants and for in vitro studies of various SBV strains.
AB - Schmallenberg virus (SBV), discovered in Germany in 2011, causes congenital malformations in ruminants. Reverse-transcription real-time PCR (RT-rtPCR) assays based on various segments of SBV have been developed for molecular detection. We developed alternative RT-rtPCR assays for SBV detection to avoid earlier reported mutations and hypervariable regions of the S and M segments of the viral genome. For SYBR Green-based detection of the S segment, the R2 value and efficiency of the developed assay were 0.99 and 99%, respectively. For probe-based S segment detection, 2 assays were developed; the first had an R2 value of 0.99 and 102% efficiency, and the second had a R2 value of 0.98 and 86% efficiency. The probe-based M segment assay had an R2 value of 1.00 and 103% efficiency. Detection limits of the RT-rtPCR assays with new primer sets were 102 and 101 copies/µL for the S and M segments, respectively. Field samples from cattle and sheep were also used for primary validation of the developed assays. Our assays should be suitable for SBV detection in ruminants and for in vitro studies of various SBV strains.
KW - M segment
KW - RT-rtPCR
KW - S segment
KW - Schmallenberg virus
KW - SYBR Green
UR - https://doi.org/10.25384/SAGE.c.5085601
U2 - 10.1177/1040638720947199
DO - 10.1177/1040638720947199
M3 - Article
C2 - 32757829
AN - SCOPUS:85089177434
SN - 1040-6387
VL - 32
SP - 710
EP - 717
JO - Journal of Veterinary Diagnostic Investigation
JF - Journal of Veterinary Diagnostic Investigation
IS - 5
ER -