Viral RNA from oral swabs was isolated with the Qiagen
MinElute virus spin kit (cat. no. 57704). Tissue RNA was extracted with RNA-STAT 60 (Tel-test”B″)/chloroform, precipitated and resuspended in
AVE Buffer (Qiagen 1020953). The qRT-PCR assay utilizes reagents that bind to a conserved region of the N gene of SARS-CoV-2. The amount of RNA was determined by O.D. reading at 260, numbers of copies were calculated and 10
8 copies per reaction were amplified. The mRNA PCR assay samples were amplified with
TaqMan RT-PCR kit (Bioline cat# BIO-78005). Serial dilutions of a standard were included. Samples were amplified in an Applied Biosystems 7500 Sequence detector at 48 °C for 30 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, and 1 min at 55 °C.
Primers/probe sequences:
2019-nCoV_N1-F:5′-GAC CCC AAA ATC AGC GAA AT-3’.
2019-nCoV_N1-R: 5′-TCT GGT TAC TGC CAG TTG AAT CTG-3’.
2019-nCoV_N1-P: 5′-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3’.
The sgmRNA assays used the
TaqMan RT-PCR kit (Bioline #BIO-78005 and was performed with an Applied Biosystems 7500 Sequence detector at 48 °C for 30 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, and 1 min at 55 °C.
Primers/probe sequences:
sg-N-F: 5′-CGATCTCTTGTAGATCTGTTCTC-3’.
sg-N-R: 5′-GGTGAACCAAGACGCAGTAT-3’.
Sg-N-P: 5′-6-FAM/TAACCAGAA/ZEN/TGGAGAACGCAGTGGG/3IABkFQ/
Hasanpourghadi M., Novikov M., Ambrose R., Chekaoui A., Newman D., Ding J., Giles-Davis W., Xiang Z., Zhou X.Y., Liu Q., Swagata K, & Ertl H.C. (2022). Heterologous chimpanzee adenovirus vector immunizations for SARS-CoV-2 spike and nucleocapsid protect hamsters against COVID-19. Microbes and Infection, 25(4), 105082.