Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA

Detection of Zoonotic Pathogens and Characterization of Novel Viruses Carried by Commensal Rattus norvegicus in New York City

“…cDNA from both strands was generated using strand-specific primers containing a tag sequence at the 5′ end (see Table S2 in the supplemental material) ( 70 ). The RNA was preheated at 70°C for 5 min with 10 pmol of specific primer and 1× reverse transcriptase buffer, followed by the addition of a preheated reaction mixture containing 1 mM MnCl 2 , 200 µM each deoxynucleoside triphosphate (dNTP), 40 U RNaseOUT, and 1 U Tth DNA polymerase (Promega).…”

Susceptibility of European jackdaws (Corvus monedula) to experimental infection with lineage 1 and 2 West Nile viruses

“…RNA was eluted in 100 μl elution buffer (Roche) and stored at −80 °C until assayed. RNA copy numbers were quantified using unmodified primers as described previously (Lim et al ., 2013a). The limit of detection of the assay was 0.95 log 10 RNA copies.…”

Schlafen 11 Restricts Flavivirus Replication

“…After optimization, the reaction was performed in a final volume of 20 l, including 10 l 2ϫ SYBR green RT-PCR mix, 0.6 l (150 nM final concentration) of forward primer 5=-CCACCGGAAGTTGAGTAGACG-3= and reverse primer 5=-TTTGCTAG CTTTAGGACCTACTATATCTACCTTTTGGTCACCCAGTCCTCCT-3=, 0.25 l iScript reverse transcriptase, and 5 l of the RNA template. These primers are specific to the 3= untranslated region (UTR) of the WNV genome and have been previously described (51). The thermal cycling conditions consisted of a 10-min reverse transcription step at 50°C and 1 min of Taq at 95°C, followed by 45 cycles of PCR consisting of denaturing at 95°C for 10 s, annealing at 65°C for 30 s, and extension at 72.0°C for 90 s, with a single step of fluorescence emission data collection followed by a final extension step for 10 min at 72°C.…”