AZURESEQ PLUS

ONE STEP UNIVERSAL RT-qPCR Kit
SARS-CoV-2, INFLUENZA A/B

Get high multiplex performance in an American-made product. Excellent sensitivity with CDC-recommended primers. All enzymes are synthesized in the United States. Fast turn-around times for customization that fits your workflow and format.

Detect SARS-CoV-2, Influenza A and Influenza B
in Less Than One Hour

Single-Tube RT-qPCR Detection

Made in America
Research Use Only

Sample Processing

Direct: Heat 100ul VTM 95C for 5’
Indirect: CDC Recommended Protocol

Probes & Primers

CDC recommended targets and sequences

Sensitivity

SARS-CoV-2
5-10 copies per reaction using CDC RNA extraction protocol, 10 copies per reaction using direct protocol.
Influenza A/B
25 copies per reaction using Direct Protocol

Amplification

40 cycles using default ramp rates
Total cycling time appx 1’10”

RT Incubation 50C 15’
Enzyme Activation 95C 2’
Amplification: 40 cycles (95C 3s, 60C 30s)

AzureSeq Plus

Multiplex Detection of
SARS-CoV-2, Influenza A and Influenza B

This multiplex RT-qPCR assay detects SARS-CoV-2, Influenza A and Influenza B on three different channels, determining if the sample contains one, two, or all three of the viruses. The assay uses RNase P as the sample control gene on the fourth channel.

Two Protocols – Same Kit

Direct: No RNA Extraction (LDT), straight from VTM/UTM*

Extracted: Use CDC Recommended RNA Extraction

Targets
N1 SARS-CoV-2, M1
Influenza A, NS2
Influenza B and human
RNaseP genes

Samples
5 ul approved VTM/UTM*
or
100 ng to 1 pg
extracted RNA (5ul)

Dyes
N1 SARS-CoV-2 = FAM
M1 FluA = Quasar 670
NS2 FluB = HEX
RNaseP = TexasRed

Parameters
Amplification 40 cycles,
LoD 5-10 copies

*Approved VTM/UTM: Copan, Puritan, CDC, Saline, PBS, MAWI EL

PERFORMANCE EVALUATION

Contrived Clinical Samples

Co-Infection Study

LoD Data Direct

LoD study for AzureSeq Plus, Direct Protocol

The inactivated virus was spiked into a negative swab sample plus saline, at 10 copies/uL, 5 copies/uL, and 2.5 copies/uL. Samples were heated at 95C for 5’, then 5uL of sample was added to a final reaction (total 20uL) containing AzureSeq Plus reagents. 20 replicates of each concentration was amplified using recommended cycling conditions (see below). The LOD for the Direct Method (no RNA purification) has been determined to be 10 copies/uL for N1 SARS-CoV-2, M1 Influenza A, and NS2 Influenza B on the BioRad CFX96 Touch Real-time Detection System at least 95% of the time using the AzureSeq Direct Plus RT-qPCR Respiratory Infection Kit with recommended cycling conditions.

Sample	N1	RNaseP	Result
CoV-1	16.08		SARS-CoV-2 Detected
CoV-2	23.29	25.15	SARS-CoV-2 Detected
CoV-3	24.96	26.51	SARS-CoV-2 Detected
CoV-4	29.93	26.32	SARS-CoV-2 Detected
CoV-5	27.54	30.66	SARS-CoV-2 Detected
CoV-6	30.12	28.95	SARS-CoV-2 Detected
CoV-7	30.07	25.31	SARS-CoV-2 Detected
CoV-8	22.82	27.28	SARS-CoV-2 Detected
CoV-9	26.26	27.73	SARS-CoV-2 Detected
CoV-10	22.91	26.26	SARS-CoV-2 Detected
CoV-11	16.12		SARS-CoV-2 Detected
CoV-12	25.19	27.08	SARS-CoV-2 Detected
CoV-13	29.79	27.66	SARS-CoV-2 Detected
CoV-14	21.01		SARS-CoV-2 Detected
CoV-15	20.19	28.65	SARS-CoV-2 Detected
CoV-16	20.12		SARS-CoV-2 Detected
CoV-17	18.16		SARS-CoV-2 Detected
CoV-18	30.26	25.12	SARS-CoV-2 Detected
CoV-19	26.99	28.13	SARS-CoV-2 Detected
CoV-20	21.93	31.44	SARS-CoV-2 Detected
CoV-21	21.36		SARS-CoV-2 Detected
CoV-22	23.26	35.61	SARS-CoV-2 Detected
CoV-23	38.41	27.43	SARS-CoV-2 Detected
CoV-24	25.65	27.31	SARS-CoV-2 Detected
CoV-25	30.5	26.69	SARS-CoV-2 Detected
CoV-26	23.86	43.88	SARS-CoV-2 Detected
CoV-27	19.78	27.3	SARS-CoV-2 Detected
CoV-28	18.81	30.02	SARS-CoV-2 Detected
CoV-29	30.22	30.07	SARS-CoV-2 Detected
CoV-30	25.46	28.73	SARS-CoV-2 Detected
CoV-31	29.19	28.26	SARS-CoV-2 Detected

31/31

SARS-CoV-2 Detection of Clinical Samples using AzureSeq Plus, Direct Protocol

Sample	FluB	RNaseP	Result
FluB-1	26.55	28.23	Influenza B Detected
FluB-2	29.19	28.99	Influenza B Detected
FluB-3	37	32.78	Influenza B Detected
FluB-4	27	29.53	Influenza B Detected
FluB-5	13.69	24	Influenza B Detected
FluB-6	23.03	22.33	Influenza B Detected
FluB-7	20.37	21.45	Influenza B Detected
FluB-8	23.48	22.13	Influenza B Detected
FluB-9	20.05	21.46	Influenza B Detected
FluB-10	21.16	22.23	Influenza B Detected
FluB-11	22.81	24.07	Influenza B Detected
FluB-12	21.88	23.63	Influenza B Detected
FluB-13	26.37	22.8	Influenza B Detected
FluB-14	19.47	21.37	Influenza B Detected
FluB-15	20.21	22.72	Influenza B Detected
FluB-16	21.59	24.83	Influenza B Detected
FluB-17	20.5	22.71	Influenza B Detected
FluB-18	17.7	21.68	Influenza B Detected
FluB-19	21.53	23.08	Influenza B Detected
FluB-20	20.69	23.16	Influenza B Detected
FluB-21	17.96	23.92	Influenza B Detected
FluB-22	24.14	26.46	Influenza B Detected
FluB-23	22.46	24.64	Influenza B Detected
FluB-24	22.89	21.29	Influenza B Detected
FluB-25	20.05	24.29	Influenza B Detected
FluB-26	21.81	27.19	Influenza B Detected

26/26

Influenza B detection of clinical samples using AzureSeq Plus, Direct Protocol

Sample	RNaseP	FluA	Result
FluA-1	26.22	28.97	Influenza A Detected
FluA-2	24.72	21.98	Influenza A Detected
FluA-3	21.14	35.33	Influenza A Detected
FluA-4	24.37	31.53	Influenza A Detected
FluA-5	22.31	23.42	Influenza A Detected
FluA-6	25.93		Not Detected
FluA-7	24.04	24.57	Influenza A Detected
FluA-8	22.31	31.24	Influenza A Detected
FluA-9	21.13	28.81	Influenza A Detected
FluA-10	21.55	22.14	Influenza A Detected
FluA-11	22.94	23.6	Influenza A Detected
FluA-12	23.41	19.36	Influenza A Detected
FluA-13	22.05	27.76	Influenza A Detected
FluA-14	22.35	29.78	Influenza A Detected
FluA-15	22.24	34.06	Influenza A Detected
FluA-17	22.65	25.1	Influenza A Detected
FluA-18	22.56	24.96	Influenza A Detected
FluA-19	24.38	24.54	Influenza A Detected
FluA-20	20.52	22.08	Influenza A Detected
FluA-21	23.49		Not Detected
FluA-22	22.82	21.73	Influenza A Detected
FluA-23	23.37		Not Detected
FluA-24	25.53		Not Detected
FluA-25	22.05	29.16	Influenza A Detected
FluA-26	24.99	21.6	Influenza A Detected
FluA-27	26.18	25.01	Influenza A Detected
FluA-28	21.22	27.84	Influenza A Detected
FluA-29	23.1	30.43	Influenza A Detected
FluA-30	22.76	23.63	Influenza A Detected
FluA-31	22.93	25.18	Influenza A Detected
FluA-32	21.35	22.88	Influenza A Detected

28/32

Influenza A detection of clinical samples using AzureSeq Plus, Direct Protocol

There was 100% correlation of the AzureSeq Plus RT-qPCR Respiratory Infection Kit to samples previously tested positive by Luminex MAGPix NxTag, Hologic Panther/ Fusion Flu A/B/RSV PCR for Influenza B and 87.5% for Influenza A. There was also 100% correlation to Zymo Quick SARS-CoV-2 kit for SARS-CoV-2 detection.

LoD Extracted

Target		20 copies/µL	10 copies/µL	5 copies/µL	1 copy/µL
CoV N1	Positive/Total	20/20	20/20	20/20	18/20
CoV N1	Ct Average	33.95	35.11	36.56	39.47
Influenza A	Positive/Total	20/20	20/20	20/20	0/0
Influenza A	Ct Average	34.22	35.59	37.12	n/a
Influenza B	Positive/Total	20/20	20/20	16/20	0/0
Influenza B	Ct Average	33.01	34.43	36.58	n/a

AzureSeq Plus Extracted Protocol LoD

Sample	N1	RNaseP	Result
CoV-1	17.55	25.16	SARS-CoV-2 Detected
CoV-2	23.48	25.68	SARS-CoV-2 Detected
CoV-3	17.84	27.21	SARS-CoV-2 Detected
CoV-4	23.36	26.78	SARS-CoV-2 Detected
CoV-5	18.05	23.35	SARS-CoV-2 Detected
CoV-6	29.65	28.38	SARS-CoV-2 Detected
CoV-7	21.72	28.14	SARS-CoV-2 Detected
CoV-8	25.43	26.06	SARS-CoV-2 Detected
CoV-9	19.77	25.7	SARS-CoV-2 Detected
CoV-10	20.47	25.16	SARS-CoV-2 Detected
CoV-11	26.89	28.08	SARS-CoV-2 Detected
CoV-12	22.06	27.18	SARS-CoV-2 Detected
CoV-13	27.06	29.51	SARS-CoV-2 Detected
CoV-14	21.36	25.14	SARS-CoV-2 Detected
CoV-15	26.54	27.63	SARS-CoV-2 Detected
CoV-16	21.46	25.88	SARS-CoV-2 Detected
CoV-17	23.09	27.46	SARS-CoV-2 Detected
CoV-18	27.69	26.28	SARS-CoV-2 Detected
CoV-19	29.8	24.83	SARS-CoV-2 Detected
CoV-20	22.79	23.19	SARS-CoV-2 Detected
CoV-21	18.81	23.96	SARS-CoV-2 Detected
CoV-22	19.46	25.6	SARS-CoV-2 Detected
CoV-23	18.71	23.74	SARS-CoV-2 Detected
CoV-24	20.16	25.78	SARS-CoV-2 Detected
CoV-25	21.95	27.33	SARS-CoV-2 Detected
CoV-26	25.67	28.56	SARS-CoV-2 Detected
CoV-27	39.21	27.35	SARS-CoV-2 Detected
CoV-28	29.11	26.08	SARS-CoV-2 Detected
CoV-29	21.31	26.08	SARS-CoV-2 Detected
CoV-30	18.83	24.97	SARS-CoV-2 Detected
CoV-31	24.35	28	SARS-CoV-2 Detected

31/31

Clinical samples AzureSeq Plus Extracted SARS-CoV-2 Results

Sample	RNaseP	FluA	Result
FluA-1	23.67	30.57	Influenza A Detected
FluA-2	21.19	22.64	Influenza A Detected
FluA-3	21.92	24.05	Influenza A Detected
FluA-4	20.82	21.23	Influenza A Detected
FluA-5	21.21	23.08	Influenza A Detected
FluA-6	27.02	31.37	Influenza A Detected
FluA-7	22.39	29.15	Influenza A Detected
FluA-8	20.51	23.34	Influenza A Detected
FluA-9	23.25	29.49	Influenza A Detected
FluA-10	23.01	21.77	Influenza A Detected
FluA-11	25.62	31.64	Influenza A Detected
FluA-12	25.02	20.6	Influenza A Detected
FluA-13	24.33	25.37	Influenza A Detected
FluA-14	27.48	24.97	Influenza A Detected
FluA-15	21.9	21.86	Influenza A Detected
FluA-16	22.82	26.79	Influenza A Detected
FluA-17	24.66	25.44	Influenza A Detected
FluA-18	22.05	27.2	Influenza A Detected
FluA-19	22.85	25.07	Influenza A Detected
FluA-20	24.46	34.89	Influenza A Detected
FluA-21	21.11	23.98	Influenza A Detected
FluA-22	24.01	25.49	Influenza A Detected
FluA-23	23.86	24.3	Influenza A Detected
FluA-24	20.38	22.06	Influenza A Detected
FluA-25	21.97	33.04	Influenza A Detected
FluA-26	24.98	34.11	Influenza A Detected
FluA-27	22.22	23.9	Influenza A Detected
FluA-28	21.5	23.28	Influenza A Detected
FluA-29	21.2	31.74	Influenza A Detected
FluA-30	21.92	26.34	Influenza A Detected
FluA-31	22.76	19.34	Influenza A Detected
FluA-32	25.44	20.83	Influenza A Detected

32/32

Clinical samples AzureSeq Plus Influenza A Results

Sample N1	FluB	RNaseP	Result
FluB-1	19.78	25.2	Influenza B Detected
FluB-2	23.91	25.87	Influenza B Detected
FluB-3	30.22	29.17	Influenza B Detected
FluB-4	19.35	24.69	Influenza B Detected
FluB-5	21.89	22.32	Influenza B Detected
FluB-6	22.85	21.82	Influenza B Detected
FluB-7	20.63	19.17	Influenza B Detected
FluB-8	23.72	20.08	Influenza B Detected
FluB-9	17.9	19.66	Influenza B Detected
FluB-10	19.29	20.02	Influenza B Detected
FluB-11	22.87	22.09	Influenza B Detected
FluB-12	24.39	21.62	Influenza B Detected
FluB-13	25.72	20.44	Influenza B Detected
FluB-14	17.97	19.01	Influenza B Detected
FluB-15	18.87	20.47	Influenza B Detected
FluB-16	20.92	24.52	Influenza B Detected
FluB-17	19.34	20.59	Influenza B Detected
FluB-18	17.25	21.34	Influenza B Detected
FluB-19	20	20.19	Influenza B Detected
FluB-20	19.07	20.82	Influenza B Detected
FluB-21	16.65	20.95	Influenza B Detected
FluB-22	21.98	25.23	Influenza B Detected
FluB-23	20.61	22.96	Influenza B Detected
FluB-24	22.06	19.06	Influenza B Detected
FluB-25	18.7	22.98	Influenza B Detected
FluB-26	19.34	26.43	Influenza B Detected

26/26

Clinical samples AzureSeq Plus Influenza B Results

There was 100% correlation of the AzureSeq Plus RT-qPCR Respiratory Infection Kit to samples previously tested positive by Luminex MAGPix NxTag, Hologic Panther/ Fusion Flu A/B/RSV PCR for Influenza A and Influenza B. There was also 100% correlation to Zymo Quick SARS-CoV-2 kit for SARS-CoV-2 detection.

ORDERING INFORMATION

SKU#	20 ul Reactions	Content
AzureSeq Plus 4-200	200	In Solution: InhibiTaq Plus, Hot Start Mastermix, RTScript, SARS CoV-2 and RNaseP primers and probes, DTT, UDG, RNase Inhibitor
AzureSeq Plus 4-1000	1000
AzureSeq Direct-2000	2000

For Research Use Only. CLIA labs must file an LDT or EUA.

Contact Us for More Information

IFUs

FAQs

How long does the cycling take?

Depending on your cycler, approximately 70′-75′.

What Dyes are used on the Primers?

For N1, FAM (absorption 493nm, emission 517nm)

For N2, HEX (absorption 533nm, emission 559nm)

For RnaseP, ROX/Tx Red (absorption 583nm, emission 603nm)

Is SeqOnce FDA registered?

Yes. Our FDA Facility Registration number is 10076042

Can you perform custom filling?

Yes, we can make AzureSeq in tube and 96/384-well format.

When will the lyophilization kit be available?

We anticipate mid-August 2020. Samples are available now for testing.
Will you submit for EUA? Yes, assuming validation data is comparable or superior to the wet version. It is currently RUO- Not For Diagnostic Use.

What order volumes can you support?

As of Jul 12, 2020 we can provide 2M reactions/week. This can be further scaled with 10 days notice. Please contact us for the most up-to-date information.

Is the reaction multiplex or multiple well single-plex?

Both are available.

What extraction kits were used for the EUA validation?

Omega Total RNA Spin Columns and KingFisher MagMax

What is the LoD?

For validation N1=5 copies/rxn, N2=10 copies/rxn.

What are the concentrations of the positive controls?

1000 cp/ul RNaseP, 200 cp/ul of N

What primer sequences are used?

https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html

How large are the amplicons?

60mer-70mer

What are the targets?

N1, N2 capsid and RP-P

What systems are AzureSeq compatible with?

It was validated using standard qPCR instruments including: QuantStudio series, BioRad FX.

Does AzureSeq have EUA?

It is EUA-Validated, not EUA-Authorized. At this time, CLIA users may choose to submit their own EUA. Upon request, we will send the FDA EUA Submission for AzureSeq.

SDS & Disclaimer

Product Disclaimer

Product Disclaimer

Category 1 CPT Code for AzureSeq Plus

87636 Infectious agent detection by nucleic acid (DNA or RNA); severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Coronavirus disease [COVID-19]) and influenza virus types A and B, multiplex amplified probe technique

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