Version June 29, 2020 

SARS-CoV-2 Synthetic RNA Fragments 

Guidance Sheet 

Research Grade Test Material ID: 10169 

Sample Description 

Each unit of RGTM 10169 consists of two tubes each containing a unique synthetic RNA 

fragment from the SARS-CoV-2 genome. Each tube contains approximately 110 μL of 

the material in a background of 5 ng/μL human Jurkat RNA (stored at -80 oC, BSL1). The concentration is approximately 5 x 106 copies/μL as estimated by multiple 

digital PCR assays (RT-dPCR). Table 1 below lists assay-specific concentration estimates 

as measured by RT-dPCR. 

Figure 1. Schematic of RGTM 10169 synthetic RNA fragments 

• Fragment 1: Total length: 3985 nt, Includes SARS-CoV-2 sequence: 25949-29698 of 

isolate USA-WA1/2020 

• Fragment 2: Total length: 3790 nt, Includes SARS-CoV-2 sequence: 12409-15962 of 

isolate USA-WA1/2020 

Purpose 

• To aid in evaluation and development of new and existing RT-qPCR assays. 

• To understand SARS-CoV-2 analytical assay performance with a synthetic RNA 

material. 

• To calibrate RT-qPCR methods and to benchmark/compare other SARS-CoV-2 

controls/materials. 

Reply Date 

Your feedback regarding use of this material will help us further test, develop, and 

improve the materials for future development. Please return your feedback survey by 
 

 

 

 

 

 

 

 

 

Version June 29, 2020 

August 31, 2020. After that date, please contact us using the information below to 

determine whether responses are still being collected and the material still stable. 

How to Reply 

A survey link will be provided on the RGTM 10169 resource page here: 

https://www.nist.gov/programs-projects/sars-cov-2-research-grade-test-material 

NIST Contacts 

Megan Cleveland Peter Vallone 

Megan.Cleveland@nist.gov Peter.Vallone@nist.gov 

Storage 

The material should be stored frozen at -80 °C. 

Notice and Warnings 

Please review the Safety Data Sheet provided with this material. The Safety Data Sheet 

can also be downloaded from: https://www.nist.gov/programs-projects/sars-cov-2-

research-grade-test-material 

Instructions for Use 

Thaw the tube at room temperature. Once thawed, vortex briefly, centrifuge briefly and 

repeat. Note: multiple freeze thaws of the material may result in lower concentration 

estimates. 

NIST Additional Information 

Supplemental data are available at: https://github.com/usnistgov/RGTM10169 

Table 1 

Median concentration values (copies/microliter) as measured by RT-dPCR assays. Note: 

“Assay Name” is an internal designation used by NIST and not intended to align nor 

conflict with alternative assay naming conventions. 

Fragment 1 Assay Name Target Region Median concentration 

(copies/microliter) 

China N N gene 2.4E+06 

Japan N gene 4.5E+06 

N1 N gene 2.2E+06 

N2 N gene 4.6E+06 

N3 N gene 4.5E+06 

Thai N gene 1.9E+06 

Sarbeco E E gene 5.4E+06 
 

 

 

 

 

Version June 29, 2020 

Fragment 2 Assay Name Target Region Median concentration 

(copies/microliter) 

China ORF1ab ORF1ab 5.2E+06 

Pasteur 1 ORF1ab 5.3E+06 

Pasteur 2 ORF1ab 5.1E+06 

RdRp ORF1ab 4.1E+05 

Tables 2 and 3 

RT-dPCR assay primers and information. The RT-dPCR assays were developed and 

optimized using primer and probe sequences from: 

https://github.com/usnistgov/RGTM10169/blob/master/whoinhouseassays_June_25_202 

0.pdf. Note: “Assay Name” is an internal designation used by NIST and not intended 

to align nor conflict with alternative assay naming conventions. 

Table 2: Fragment 1 

Assay 

Developer 

Assay 

Name 

Primer and Probe Sequences Alternate assay 

names 

Centers for 

Disease 

Control, China 

China N F- ggggaacttctcctgctagaat 

P- ttgctgctgcttgacagatt 

R- cagacattttgctctcaagctg 

Centers for Disease 

Control, China, novel 

coronavirus Target 2 

National 

Institute of 

Infectious 

Diseases, 

Japan 

Japan F- aaattttggggaccaggaac 

P- atgtcgcgcattggcatgga 

R- tggcagctgtgtaggtcaac 

National Institute of 

Infectious Diseases, 

Japan, NIID_2019-

nCoV_N 

United States 

Centers for 

Disease 

Control 

N1 F- gaccccaaaatcagcgaaat 

P- accccgcattacgtttggtggacc 

R- tctggttactgccagttgaatctg 

United States Centers 

for Disease Control, 

2019-nCoV N1 

United States 

Centers for 

Disease 

Control 

N2 F- ttacaaacattggccgcaaa 

P- acaatttgcccccagcgcttcag 

R- gcgcgacattccgaagaa 

United States Centers 

for Disease Control, 

2019-nCoV N2 

United States 

Centers for 

Disease 

Control 

N3 F- gggagccttgaatacaccaaaa 

P- aycacattggcacccgcaatcctg 

R- tgtagcacgattgcagcattg 

United States Centers 

for Disease Control, 

2019-nCoV N3 
 

 

Version June 29, 2020 

World Health 

Organization 

Sarbeco E F- acaggtacgttaatagttaatagcgt 

P- acactagccatccttactgcgcttcg 

R- atattgcagcagtacgcacaca 

World Health 

Organization, n-CoV 

Sarbeco-E 

Ministry of 

Public Health, 

Thailand 

Thai F- cgtttggtggaccctcagat 

P- caactggcagtaacca 

R- ccccactgcgttctccatt 

Ministry of Public 

Health, Thailand , 

2019-nCoV 

Table 3: Fragment 2 

Assay 

Developer 

Assay 

Name 

Primer and Probe Sequences Alternate assay 

names 

Centers for 

Disease 

Control, China 

China 

ORF1ab 

F- ccctgtgggttttacacttaa 

P- ccgtctgcggtatgtggaaaggttatgg 

R- acgattgtgcatcagctga 

Centers for 

Disease Control, 

China, novel 

coronavirus 

Target 1 

Pasteur 

Institute 

Pasteur 1 F- atgagcttagtcctgttg 

P- agatgtcttgtgctgccggta 

R- ctccctttgttgtgttgt 

Pasteur Institute, 

COVID-19 Target 

1, IP2 

Pasteur 

Institute 

Pasteur 2 F- ggtaactggtatgatttcg 

P- tcatacaaaccacgccagg 

R- ctggtcaaggttaatatagg 

Pasteur Institute, 

COVID-19 Target 

2, IP4 

World Health 

Organization 

RdRp F- gtgaratggtcatgtgtggcgg 

P- caggtggaacctcatcaggagatgc 

R- caratgttaaasacactattagcata 

World Health 

Organization, nCoV RdRp