Skip to main content

Table 2 Published primers and nested RT-PCR conditions

From: Evaluation on the use of Nanopore sequencing for direct characterization of coronaviruses from respiratory specimens, and a study on emerging missense mutations in partial RdRP gene of SARS-CoV-2

Primer sequences
1st PCR
Forward 5′-GGN TGG GAY TAY CCN AAR TGY GA-3′ 760 bp amplicon
Reverse 5′-RHG GRT ANG CRT CWA TDG C-3′
2nd PCR
Forward 5′-GGT TGG GAC TAT CCT AAG TGT GA-3′ 440 bp amplicon
Reverse 5′-CCA TCA TCA GAT AGA ATC ATC AT-3′
Reverse transcription & First PCR
Master mix constituents per reaction PCR profile
5× RT buffer 5 µL    
dNTP (10 mM) 1 µL 45 °C 30 m  
5× Q solution 5 µL 95 °C 15 m  
Forward primer (200 µM) 0.5 µL 94 °C 30 s  
Reverse primer (200 µM) 0.5 µL 45 °C 30 s 50×
One-step enzyme mix 1 µL 72 °C 1 m  
RNase-free water 2 µL 72 °C 10 m  
RNA 10 µL 15 °C Hold  
Total 25 µL    
Second PCR
Master mix constituents per reaction PCR profile
10x PCR buffer 5 µL    
MgCl2 (25 mM) 3 µL 95 °C 10 m  
dNTP (10 mM) 1 µL 95 °C 15 s  
Forward primer (10 µM) 1 µL 45 °C 30 s 35×
Reverse primer (10 µM) 1 µL 72 °C 30 s  
PCR-grade water 33.5 µL 72 °C 5 m  
AmpliTaq Gold 0.5 µL 15 °C Hold  
Purified 1st PCR amplicon 5 µL    
Total 50 µL