Comparison of National RT-PCR Primers, Probes,

and Protocols for SARS-CoV-2 Diagnostics April 13, 2020

In this fact sheet, we provide a detailed comparison of several many different protocols for reverse transcriptase polymerase
RT-PCR tests developed by various countries. Our review is chain reaction (RT-PCR) diagnostic testing for SARS-CoV-2,
limited to those listed in the World Health Organization (WHO) the causative agent of COVID-19 disease, they are not all
resource of in-house–developed molecular assays (accessible designed to target the same segments of the genome (see
here) that have been used as the national test of choice in many Figure 1). Many nationally used RT-PCR protocols target the
countries and in regional reference laboratories. Depending on nucleocapsid phosphoprotein (N protein) of SARS-CoV-2. This
the test protocol, various primers, probes, concentrations of is because, among other reasons, it is a highly conserved region
primers and probes, recommended extraction methods, and RT- in coronaviruses and likely to deliver consistent results as the
PCR kits are used. pandemic progresses.

Primers are small fragments of single-stranded DNA designed The N region genome map in Figure 2 below includes primers
to bind specifically to a single region in the genome to allow from 5 institutions that had at least 1 target within the N
for precise amplification of the diagnostic target area. Probes protein. As such, this is not an exhaustive look at all primers in
bind to the target area and give off a fluorescent signal as use for RT-PCR diagnostics of COVID-19; however, it does
amplification of that area increases. This fluorescent signal is illustrate the variability in the primer selection process between
then read by the quantitative real-time PCR machine where the different institutions, even when creating primers for the same
reaction is occurring to give a diagnostic read-out. Molecular genomic region of the same virus.
scientists use many different tools to design the most appropriate
primers to create functional diagnostic targets. While there are

Figure 1. SARS-CoV-2 genome (NC_004718.3). Orf, open reading frame; S, spike gene; E, envelope gene; M, membrane gene; N,
nucleocapsid gene. Gene size drawn to scale.

How to read the N region genome map: How to interpret sensitivity and specificity
• Different colors represent primer sets from different measures in the RT-PCR Master Table
protocols. In the case of the United States, there are 2 In each set of primers described, sensitivity and specificity are
separate primer pairs, all in the same color. included. Sensitivity and specificity measures were not available
• Primer pairs include a forward primer and a reverse primer for all protocols.
in order to read both strands of DNA. Within the same
protocol, the first color-blocked sequence is the forward Sensitivity refers to the level at which the protocol can recognize
primer, the intervening uncolored sequence is the area the presence of SARS-CoV-2 RNA. The higher the sensitivity,
being amplified during PCR, and the second corresponding the less viral RNA is required for a positive test result. Tests
color-blocked sequence is the reverse primer. with high sensitivity have a lower chance of missing a true
• Note: Although SARS-CoV-2 is an RNA virus, positive (ie, declaring a false negative), an important factor
during the RT-PCR process it is reverse-transcribed when testing positive or negative can affect treatment and
into DNA for increased stability and ease of use. hospitalization decisions. Primer sets and probes can be tested
• Mixed colors denote areas of primer overlap between for their sensitivity using a range of fixed concentrations of in
national protocols. For example, the forward primer from vitro transcribed SARS-CoV2- viral RNA transcripts (positive
Thailand’s protocol overlaps slightly with the reverse N1 controls). The limit of detection (LOD) is the lowest possible
primer from the United States protocol. concentration of SARS-CoV-2 that can be detected under the
experimental conditions in at least 95% of all reactions. Most
tests aim for an LOD of 10 RNA copies per reaction. A test with
a low LOD would be considered more sensitive. Having a low
LOD means that the test can detect low-level infections.

© Johns Hopkins Center for Health Security, centerforhealthsecurity.org 04/13/2020

Fact Sheet: Comparison of National RT-PCR Primers, Probes, and Protocols for SARS-CoV-2 Diagnostics 2

Specificity refers to whether the test recognizes only SARS-

CoV-2 RNA and not other closely related pathogens or human
RNA transcripts. Tests with high specificity have a lower chance
of declaring a false positive; rather, tests with high specificity
have a greater ability to rule out true negatives from further
clinical suspicion. Primer and probe specificity can be increased
by ensuring they do not share great sequence similarity with
other viral RNA sequences. Choosing primers and probes that
recognize highly specific genome domains of SARS-CoV-2,
such as the N domain, can help to increase specificity. Most
laboratories have also assessed test specificity by checking
for cross-reactivity with a panel of respiratory viruses, such
as influenza A (H1N1), influenza A (H1N3), influenza B,
rhinovirus, and other human coronaviruses. Ideally, cross-
reactivity should be tested on RNA derived from patient samples.
To be deemed adequately specific, the test should not read
positive for any virus other than SARS-CoV-2. 

The master table is not intended to make claims on which test

is better or more reliable than others; more research would be
required to answer those questions. Still, it is important to be
able to understand what parameters the developing institutions
used when validating their protocols.

© Johns Hopkins Center for Health Security, centerforhealthsecurity.org 04/13/2020

 Fact Sheet: Comparison of National RT-PCR Primers, Probes, and Protocols for SARS-CoV-2 Diagnostics 3

Note: The beginning and ending nucleotides represented on this page do not represent the actual boundaries of the N protein but were rather arbitrary points chosen
for ease of visibility of the highlighted SARS-CoV-2 primers. The full sequence of the N protein can be accessed here.

GATATCGGTAATTATACAGTTTCCTGTTCACCTTTTACAATTAATTGCCAGGAACCTAAATTGGGTAGTCTTGTAGTGCGTTGTTCGT
TCTATGAAGACTTTTTAGAGTATCATGACGTTCGTGTTGTTTTAGATTTCATCTAAACGAACAAACTAAAATGTCTGATAATGGACCC US, N1
CAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGC
GCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTT
AAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGA
ATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCC
CTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCC
TGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCA
GTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTA
GAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAA
GGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAA
GCATACAATGTAACACAAGCTTTTGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGG
AACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAG US, N2
TCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGC
TGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGA

Developed in Institution F Primer 5′ – 3′ R Primer, 5′ – 3′ Amplicon Size

(reverse compliment of below sequences highlighted in map)
United States US CDC N1: GACCCCAAAATCAGCGAAAT N1: TCTGGTTACTGCCAGTTGAATCTG N1: 71 bp
N2: TTACAAACATTGGCCGCAAA N2: GCGCGACATTCCGAAGAA N2: 67 bp
Thailand National Institute of
CGTTTGGTGGACCCTCAGAT CCCCACTGCGTTCTCCATT 57 bp
Health
China China CDC GGGGAACTTCTCCTGCTAGAAT CAGACATTTTGCTCTCAAGCTG 99 bp
Hong Kong University of Hong
Kong, Li Ka Shing TAATCAGACAAGGAACTGATTA CGAAGGTGTGACTTCCATG 110 bp
School of Medicine
Japan National Institute of
AATTTTGGGGACCAGGAAC TGGCAGCTGTGTAGGTCAAC 155 bp
Infectious Diseases
Primers source: https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf?sfvrsn=de3a76aa_2
Figure 2. N region genome map and abridged table display the location of national primers and their true distance from each other in the
SARS-CoV-2 genome.

© Johns Hopkins Center for Health Security, centerforhealthsecurity.org 04/13/2020

 Fact Sheet: Comparison of National RT-PCR Primers, Probes, and Protocols for SARS-CoV-2 Diagnostics 4

Master Table of national RT-PCR primers, probes, and protocol specifications (where available). Source: https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf?sfvrsn=de3a76aa_2
Amplicon Concentration/ Does the protocol
Primer Primers (sequence) Primers (sequence) Primers (sequence) Size (base Volume of recommend specific
Country Target(s) Forward 5' -3' Reverse 5' -3' Probe 5'-3' pairs) Sensitivity Specificity Reagents kits?
N1 GACCCCAAAATCAGCG TCTGGTTACTGCCAGTT FAM- 71 bp Limit of detection Probe showed high 20 μM primers, 5 For the RT-qPCR, they
AAAT GAATCTG ACCCCGCATTACGTTTGGTG (LOD): 10^0.5 RNA sequence homology μM probe; 15 μL recommend TaqPath™ 1-
GACC-BHQ1 copies/ul for Qiagen with SARS coronavirus total volume Step RT-qPCR Master Mix.
EZ1 Advanced XL Kit and Bat Sars-like For extraction, they
recommend bioMérieux
and 10 RNA copies/ul coronavirus. F and R
NucliSens® systems,
for Qiagen DSP Viral primer showed no QIAamp® Viral RNA Mini
Mini Kit. 100% primer sequence homology Kit, QIAamp® MinElute
alignment with all with SARS coronavirus Virus Spin Kit or RNeasy®
available 2019-nCov or Bat Sars-like Mini Kit (QIAGEN), EZ1
N2 TTACAAACATTGGCCG GCGCGACATTCCGAAGA FAM- 67 bp sequences, 1 mismatch coronavirus. F primer DSP Virus Kit (QIAGEN),
USA CAAA A ACAATTTGCCCCCAGCGCTT with the N1 F primer showed high sequence Roche MagNA Pure
(CDC) CAG-BHQ1
with 1 deposited homology to Bat SARS- Compact RNA Isolation Kit,
sequence, but impact like coronavirus. R Roche MagNA Pure
will be negligible, primer and probe Compact Nucleic Acid
Isolation Kit, and
primers should still showed no significant
Roche MagNA Pure 96 DNA
N3 GGGAGCCTTGAATAC TGTAGCACGATTGCAGC FAM- 72 bp bind. homology with human and Viral NA Small Volume
(removed ACCAAAA ATTG AYCACATTGGCACCCGCAAT genome, other Kit, and Invitrogen
from CCTG-BHQ1 coronaviruses, or ChargeSwitch®, Total RNA
diagnostic human microflora. Cell Kit.
panel
3/15/20)
Orf1ab CCCTGTGGGTTTTACA ACGATTGTGCATCAGCT FAM- Not stated Not stated Not stated Not stated
CTTAA GA CCGTCTGCGGTATGTGGAA —
China AGGTTATGG-BHQ1
(CDC) N GGGGAACTTCTCCTGC CAGACATTTTGCTCTCA FAM- Not stated Not stated Not stated Not stated
TAGAAT AGCTG TTGCTGCTGCTTGACAGATT —
-TAMRA
nCoV_IP2 ATGAGCTTAGTCCTGT CTCCCTTTGTTGTGTTG Hex- 108 bp 95% hit rate for approx. No cross-reactivity with Final concentration RNA extraction via
TG T AGATGTCTTGTGCTGCCGG 100 copies of RNA other respiratory viruses of 0.4 μM of each NucleoSpin Dx Virus
TA-BHQ1 (Macherey Nagel 740895.50)

© Johns Hopkins Center for Health Security, centerforhealthsecurity.org 04/13/2020

 Fact Sheet: Comparison of National RT-PCR Primers, Probes, and Protocols for SARS-CoV-2 Diagnostics 5

nCoV_IP4 GGTAACTGGTATGATT CTGGTCAAGGTTAATAT FAM- 107 bp genome equivalent (influenza A, H3N2, primer and 0.2 μM Invitrogen SuperscriptTM III
TCG AGG
TCATACAAACCACGCCAGG- (LOD) for 1E7 RNA etc) of probe Platinum® One-Step qRT-
BHQ1 copies of transcript, it's PCR system (ref: 11732-088)
France E ACAGGTACGTTAATAG ATATTGCAGCAGTACGC FAM- 125 bp ~21 cylces, for 1E4 it’s
(Institut gene/E_Sa TTAATAGCGT ACACA ACACTAGCCATCCTTACTGC ~30 cycles
Pasteur) rbeco GCTTCG-BHQ1
1 ul of 20 xprimer RNA extracted using
Japan and probe mix in a QIAamp virual RNA mini kit
Average Cq value of
(National (Qiagen). Reverse
20 ul reaction with 5
FAM- specimen was 36.7 and
Institute AAATTTTGGGGACCA TGGCAGCTGTGTAGGT ul of RNA. F primertranscription via Super Script
N ATGTCGCGCATTGGCATGG — 35.0 for the positive Not stated
of GGAAC CAAC
A-BHQ at 500 nM, R primerIV Reverse Transcriptase
control (500 copies of (Thermo). RT-PCR via
Infectious at 700 nM, probe at
RNA transcript) QuantiTect Probe RT-PCR
Disease) 200 nM. Kit (Qiagen).
RdRP GTGARATGGTCATGT CARATGTTAAASACACTA P1: FAM- LOD: 3.8 RNA No nonspecific RdRP: F-600 RNA extracted using MagNA
GTGGCGG TTAGCATA CCAGGTGGWACRTCATCMG copies/rxn, 95% hit reactivity with water nM/reaction, R-800 Pure 96 system (Roche), RT-
GTGATGC-BBQ, P2: FAM- rate; 95% CI: 2.7-7.6 samples detected. No nM/rxn, P-100 nM PCR via Superscript III one
CAGGTGGAACCTCATCAGG RNA copies/reaction reactivity with other each/rxn, step RT-PCR ystem with
AGATGC-BBQ — Platinum Taq Polymerase
human respiratory
(Invitrogen).
Germany viruses and bacteria
(Charité) (used RNA from
patient samples).
E gene ACAGGTACGTTAATAG ATATTGCAGCAGTACGC FAM- LOD: 5.2 RNA E gene: F-400
TTAATAGCGT ACACA ACACTAGCCATCCTTACTGC copies/reaction, at 95% nM/rxn, R-400
GCTTCG-BBQ —
hit rate; CI: 3.7-9.6 nM/rxn, P-200
RNA copies/reaction nM/rxn
Orf1b- TGGGGYTTTACRGGT AACRCGCTTAACAAAGC FAM- 132 bp Not stated No reactivity with 10 μM primers, 10 QIAamp Viral RNA Mini Kit
nsp14 AACCT ACTC TAGTTGTGATGCWATCATG respiratory cultured μM probes (QIAGEN, Cat#52906) or
Hong ACTAG-TAMRA viruses and clinical equivalent
Kong N TAATCAGACAAGGAAC CGAAGGTGTGACTTCCA FAM- 110 bp Not stated samples. and TaqMan Fast Virus
TGATTA TG GCAAATTGTGCAATTTGCG Master mix.
G-TAMRA
Macherey-Nagel Nucleospin
Positive control RNA virus (Cat. No 740956) and
CGTTTGGTGGACCCTC CCCCACTGCGTTCTCCAT FAM- 40 μM primers, 10 Invitrogen superscriptTM III
Thailand N — detected at less than 38 Not stated
AGAT T CAACTGGCAGTAACCABQH1 μM probe Platinum One-Step Quantitative
cycles. (Cat No.
11732-020 or 11732-088)

© Johns Hopkins Center for Health Security, centerforhealthsecurity.org 04/13/2020