Please find attached instructions for the assemble of a fast and cheaper version of RT-qPCR kit.
The kit got the approval from the CDC of Shenyang city and the CDC from Liaoning Provence in China, and it has been in use extensively in China and other countries. The EU CE application on the way. This is part of the humanity help that Xiushan Yin and his team at Biotech (http://www.bio2k.cn , only in Chinese sorry for the moment) have been donating to many countries.
In parallel we have developed a LAMP kit for places without access to qPCR (MedRxiv link).
Brochure and example case: RT-qPCR kit for SARS-CoV-2 detection-Biotech
Manual: The Instructions of Real Time PCR Detection Kit
Kit content to assemble by:
| label | sequence | fragment length(bp) | production lengeh(bp) | site | gene | primer final concentration |
| BIO-ORF-F | 5′-CCCTGTGGGTTTTACACTTAA-3′ | 21 | 119 | 13342-13460 | ORF1ab | 2μM |
| BIO-ORF-R | 5′-ACGATTGTGCATCAGCTGA-3′ | 19 | ORF1ab | 2μM | ||
| BIO-ORF-P | 5′-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3′ | 28 | 28 | ORF1ab | 0.5μM | |
| BIO-N-F | 5′-GGG AGC CTT GAA TAC ACC AAA A-3′ | 22 | 72 | 28680-28752 | structural protein N | 2μM |
| BIO-N-R | 5′-TGT AGC ACG ATT GCA GCA TTG-3′ | 21 | structural protein N | 2μM | ||
| BIO-N-P | 5′-HEX-AYC ACA TTG GCA CCC GCA ATC CTG-BHQ2-3′ | 24 | 24 | structural protein N | 0.5μM |
The 650 µl reaction solution is for 50 tests. We recommend using 40 µl of enzyme mix from ThermoFisher (12574026) and 500 µl 2*buffer (from thermo 12574026) with 110 µl DEPC H2O [no need for extra MgSO4].
Update (21 March 2020):
Potential to mix with HUDSON to avoid RNA extraction, although decreases a bit sensitivity.
HUDSON (heating unpurified diagnostic samples to obliterate nucleases)
For the HUDSON protocol, cell culture supernatants were harvested, placed on ice, and mixed with tris(2-carboxyethyl)phosphine (TCEP, Thermo Fisher Scientific, used at 100 mM final concentration) and EDTA (Thermo Fisher Scientific, 1 mM final concentration). HUDSON mixtures were then incubated at 50C for 5 min, followed by 95C for 5 min to inactivate nucleases and viral particles respectively. Heat inactivated products were not diluted, but HUDSON products were diluted 1:10 with nuclease-free water prior to RT-qPCR. RT-qPCR was performed using the Power SYBR. It comes originally from this paper https://www.ncbi.nlm.nih.gov/pubmed/29700266