Table 5.

RT-PCR reaction conditions of the available studies.

Reference The papers marked with an asterisk (*) were not certified by peer review (preprints)	Location given in alphabetical order	Template volume used	Reaction mixture volume	Selected primers	PCR reaction Mix	Instrumentation	Cycling parameters	Quality control measures	Other analyses
[34]	Australia	6 μL	40 μL	1N_Sarbeco 2NIID_2019-nCOV_N	iTaq Universal Probes One-Step Reaction Mix	BioRad CFX 96 thermal cycler	N_Sarbeco 10 min at 50 °C 3 min at 95 °C X 45 cycles of: 15 sec at 95 °C 30 sec at 58 °C NIID_2019-nCOV_N 10 min at 50 °C 15 min at 95 °C X 45 cycles of: 15 sec at 95 °C 60 sec at 60 °C	•gBlocks gene fragments as + ve controls and standards •RNA inhibition determination through Sketa22 (Ochorhynchus keta) RT-qPCR assay •Reagent blanks •Extraction blanks •Separate laboratories for RNA extraction and RT-qPCR analyses •LOD of N_Sarbeco: 8.3 copies/reaction •LOD of NIID_2019-nCOV_N: not known	Sequencing using Sanger and Illumina sequencing platforms
[35]	Australia	5 μL	25 μL	MHV virus	iTaq Universal Probes One-Step Reaction Mix	BioRad CFX 96 thermal cycler	10 min at 50 °C 5 min at 95 °C X 40 cycles of: 15 sec at 95 °C 60 sec at 60 °C	•gBlocks gene fragments as + ve controls and standards •Negative controls •RNA inhibition determination through Sketa22 (Ochorhynchus keta) RT-qPCR assay •Reagent blanks •Extraction blanks •Separate laboratories for RNA extraction and RT-qPCR analyses	Not reported
[4]	Australia	3 μL	20 μL	CDC N1, N2 and E NIID_2019-nCoV N assay	iTaq Universal Probes One-Step Reaction Mix	BioRad CFX 96 thermal cycler	Not reported	•gBlocks gene fragments as + ve controls and standards •Negative controls •RNA inhibition determination through Sketa22 (Ochorhynchus keta) RT-qPCR assay •Reagent blanks •Extraction blanks •Separate laboratories for RNA extraction and RT-qPCR analyses	•RT-ddPCR analysis •NextSeq Illumina sequencing
[7]*	Brazil	Not reported	Not reported	N1, RdRp, S	OneStep qPCR Quantinova kit	Not reported	Not reported	Murine Norovirus	Not reported
[25]*	France	Not reported	Not reported	N1 and N3	Not reported	Not reported	Not reported	VP40-encoding RNA of Ebola virus as normalisation standard	Not reported
[6]*	France	5 μL	25 μL	E and RdRp	Fast virus 1-step Master mix	Viaa7 Real Time PCR System	10 min at 55 °C 3 min at 95 °C X 45 cycles of: 15 sec at 95 °C 58 sec at 30 °C	Internal positive control	Not reported
[32]*	India	Not reported	Not reported	ORF1ab, N, S	TaqPath Covid-19 RT-PCR Kit	Not reported	Not reported	Positive controls Negative controls	Not reported
[27]*	Israel	Not reported	Not reported	E gene	Fast Start Universal Probe Master	Step One Plus real -time PCR system	Not reported	•Positive controls •Negative controls	Not reported
[22]	Italy	2.5 μL 5 μL of first PCR product	25 μL 50 μL	ORF1ab (broad range and SARS-CoV-2 specific) and NIID_WH-1	•Kit Platinum SuperFit Green PCR Master Mix •DreamTaq Polymerase and buffer	Not reported	30 sec at 98 °C X 35 cycles of: 10 sec at 95 °C 10 sec at 50 °C 10 sec at 54 °C 30 sec at 72 °C 5 min at 72 °C 30 sec at 98 °C X 45 cycles of: 10 sec at 95 °C 10 sec at 50 °C 10 sec at 54 °C 30 sec at 72 °C 5 min at 72 °C	Negative controls	Sequencing
[24]*	Italy	2 μL and 5 μL	25 μL	ORF1ab, E and RdRp	•Kit Platinum SuperFit Green PCR Master Mix •Ultrasense one-step qRT-PCR System •AgPath-ID One-Step RT-PCR system	Not reported	30 sec at 98 °C X 35 cycles of: 10 sec at 98 °C 10 sec at 54 °C 30 sec at 72 °C 5 min at 72 °C 30 min at 50 °C X 45 cycles of: 15 sec at 95 °C 60 or 95 sec at 58 °C 30 sec at 60 °C	•Positive synthetic controls •Negative controls	Sequencing
[9]*	Italy	2.5 μL	Not reported	N1, N2, N3, ORF1ab, E	Not reported	Not reported	Not reported	Not reported	•Whole genome sequencing •Genome assembly and phylogenomic analysis •Cell culture and virus isolation
[33]*	Japan	2.5 μL	25 μL	N_Sarbeco and NIID_2019-nCOV_N, N1, N2	Probe qPCR Mix with UNG	Dice Real Time System TP800	10 min at 25 °C 30 sec at 95 °C X 45 cycles of: 5 sec at 95 °C 60 sec at 60 °C	•Non-inhibitory control •Pepper mild mottle virus control •Negative controls •GBlocks gene fragments containing amplification region sequences used to generate standard curve	Not reported
[26]	Spain	Not reported	25 μL	N1, N2, N3	OneStep PrimeScript RT-PCR kit	LightCycler 480 system	10 min at 50 °C 3 min at 95 °C X 45 cycles of: 3 sec at 95 °C 30 sec at 55 °C	•Positive control •Negative controls	Not reported
[21]* [38]	The Netherlands	5 μL	20 μL	N1, N2, N3 and E	TaqMan Fast Virus 1-step Master Mix	BioRad CFX 96 thermal cycler	5 min at 50 °C X 45 cycles of: 10 sec at 95 °C 30 sec at 60 °C	•Positive controls •Negative controls •In-vitro synthesized RNA from denge virus type 2	Not reported
[28]*	Turkey	5 μL	20 μL	RdRp	QuantiNova Pathogen + IC Kit	BioRad CFX 96 thermal cycler	Not reported	•Internal controls	Not reported
[29]*	USA	2.5 μL	25 μL	IP2 and IP4 of RdRp	Reliance One-Step Multiplex RT-qPCR Supermix	QuantStudio3 or QuantStudio5	10 min at 50 °C 10 min at 95 °C X 45 cycles of: 10 sec at 95 °C 30 sec at 59 °C	•CrAssphage spiked samples	Not reported
[30]	USA	2.5 μL	25 μL	N1, N2	PerfecTa qPCR ToughMix	BioRad CFX 96 thermal cycler	10 min at 95 °C X 45 cycles of: 10 sec at 95 °C 30 sec at 55 °C	• Pseudomonas bacteriophage Φ6	Not reported
[31]*	USA	Not reported	Not reported	N1, N2, N3	Not reported	Not reported	Not reported	•N1, N2, N3 positive controls	Sanger sequencing
[8]*	USA	5 μL	40 μL	2019 nCoV CDC EUA Kit (N1 and N2)	TaqPath 1-step RT-qPCR Master Mix	ABI 7500 Fast Real-Time PCR system	2 min at 25 °C 15 min at 50 °C 2 min at 2 °C X 45 cycles of: 3 sec at 95 °C 30 sec at 55 °C	•Negative controls •Triplicate measurements	Sanger Sequencing