COVID-19

Reagent Solutions for Molecular and Immunological SARS-CoV-2 Diagnostic Assays.

Reagent Solutions for Molecular and Immunological SARS-CoV-2 Diagnostic Assays COVID-19

The key to fighting the COVID-19 pandemic is through diagnostic testing. By identifying infectious individuals, it is possible to trace the pathogen’s spread and stop the chain of transmission. However, the SARS-CoV-2 virus has mutated over time, resulting in genetic variation in the population of circulating viral strains over the course of the COVID-19 pandemic. Viral mutations can impact the performance of a PCR, antigen, or serology test, causing false negative results. Molecular tests that use multiple genetic targets to determine a final result and rapid tests that detect the highly conserved nucleocapsid protein are less likely to be impacted by the increased prevalence of genetic variants. Due to the inherent design differences of each test, mutations can affect each assay differently.

PCR Assays Considered the gold standard for COVID-19 testing and mainly uses nasopharyngeal or nasal swabs to detect SARS-CoV-2 viral RNA. The most common tested gene targets include the E, S, and N genes and the open reading frame ORF1a/1b of SARS-CoV-2.

Rapid Antigen Assays Lateral flow-based assays which detect antigen viral proteins from SARS-CoV-2, most commonly the nucleocapsid protein (predominant antigenic protein and the most abundant shed antigen throughout the SARS-CoV infection). Test can be self administered and rely on nasopharyngeal, nasal or saliva swabs.

Serology Tests These tests are designed to detect antibody responses developed against the viral antigen and generally detect IgM and IgG antibodies against SARS-CoV-2. Structural alterations in antibodies as a result of mutations influence the binding efficiency to the target regions.

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Product Selection Chart

Inhibitor-Tolerant Mixes Optimized for extraction-free assays from crude clinical specimens (sputum, saliva and stool)

Fast Mixes Ideal for multiplex assays on fast, automated high-throughput systems

Lyo-ready Mixes

Air-Dryable Mixes Specimen-specific mixes (saliva, blood, urine, stool) designed for direct detection from clinical samples and compatible for creating ambient- temperature stable assays using air-drying

Ready-to-use mix compatible with

lyophilization protocols to produce an ambient temperature stable assay

Features

Concentration

Master Mix

Antibody

Hot-Start

Ambient- Temperature Assays

Multiplex Reactions

RNA/DNA Extraction-Free Protocols

Inhibitor-Tolerant

RT-qPCR Extraction Control • Monitors assay inhibition

VLP RNA Extraction Control

• Closely mimics the test sample • Undergoes the same processing from lysis and extraction to detection • Compatible with lyophilization for creating freeze-dried mixes

• Suitable for use with inhibitor-rich samples

MDX068 (Red, Cy5) MDX069 (Orange, HEX) MDX071 (Custom)

MDX028 (Red, Quasar 670) MDX029 (Orange, Cal Fluor Orange)

Recommended

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Molecular Reagents

Optimized RT-qPCR Mixes

Meridian’s portfolio of RT-qPCR master mixes have been designed to simplify the development of SARS-CoV-2 molecular assays and enable new features such as extraction-free amplification. Each master mix contains a hot-start polymerase, dNTPs, buffer and other components optimized for each particular application (e.g. lyophilization). Only primers and probes are required to complete the assay formula. SARS-CoV-2 requires an RNA extraction step which is time limiting and can result in RNA being lost during the extraction process. However, extraction is typically required to remove inhibitors which would otherwise impact an assay’s sensitivity and accuracy. In order to address this challenge, Meridian has developed several inhibitor-tolerant mixes capable of delivering sensitive multiplex detection, even in the presence of difficult inhibitors found in sputum, saliva and stool specimens. This new mix offers a novel alternative to nucleic acids extraction, saving both time and labor which are vitally important in viral screening assays that require rapid detection for infection control.

DNA/RNA purification Add to master mix

Sample

Go to PCR

Conventional approach

Meridian’s Direct Solution

Sample

Go to PCR

No DNA/RNA extraction needed

Specimen-Specific Sample Mix

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RT-qPCR Master Mixes

Inhibitor-Tolerant RT-PCR Mix

PRODUCT HIGHLIGHTS

PRODUCT

CAT NO. VOLUME REACTIONS

• Designed for amplification from crude samples such as blood, urine, cerebral spinal fluid (CSF) and milk • 4x concentrated - ideal for developing qualitative assays where sample is limited • Sensitive amplification in singleplex and multiplex assays

5 mL

500 Rxns

Inhibitor-Tolerant qPCR Mix

MDX013

100 mL 10,000 Rxns

5 mL

100 Rxns

Inhibitor-Tolerant RT-qPCR Mix

MDX016

100 mL 10,000 Rxns

Exhibits a high tolerance to PCR inhibitors from clinical samples

Sputum Specimens Amplification profile of inactivated influenza virus spiked into samples containing 5% sputum or no sputum. The data illustrates that the performance of Inhibitor-Tolerant RT-qPCR Mix (MDX016) in the presence of 5% artificial sputum ( red ) is the same Fast One-Step RT-qPCR Mix (MDX032) with no sputum ( blue ). In contrast, the sensitivity and performance of Fast One-Step RT-qPCR Mix (MDX032) significantly decreases in the presence of 5% artificial sputum ( black ) compared to no sputum or compared to Inhibitor-Tolerant RT-qPCR Mix (MDX016) with sputum.

MDX016 (Artificial sputum) | MDX032 (No sputum) | MDX032 (Artificial sputum)

Saliva Specimens MDX016 ( red ) and MDX032 ( black ) amplification traces of Influenza A spike in presence of saliva swabs (COPAN ESwab 359C) at 10% (left) and 20% (right) final concentration. With earlier Ct (approx. 4 Ct) and higher fluorescence (approx. +50%), the results demonstrate the superiority of Inhibitor-Tolerant RT-qPCR Mix (MDX016) against a standard RT-qPCR Mix (MDX032) for the detection of viral RNA in presence of saliva swab resuspension in UTM.

10% Saliva

20% Saliva

MDX016 | MDX032

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RT-qPCR Master Mixes

Air-Dryable TM 1-Step RT-qPCR Mix PRODUCT HIGHLIGHTS • Simple and easy to use • Inhibitor-tolerant mix designed singleplex or multiplex detection from crude lysates • Can be used in a wet or dry format, and compatible with a range of air-drying protocols to produce an ambient temperature stable mix • Compatible with a range of air-drying protocols to produce an ambient temperature stable mix High-tolerance to PCR inhibitors in sputum samples Amplification profile of a mouse RNA target spiked into samples containing 10%, 5% or 0% artificial sputum in Universal Transport Media (UTM). The data illustrates that the performance of Air-Dryable 1-Step RT-qPCR Mix (MDX095) exhibits high tolerance towards inhibitors present in artificial sputum and UTM. Air-Dryable TM RNA/DNA LAMP Mix PRODUCT HIGHLIGHTS • Fast reaction kinetics – results in half the time • Complete, pre-formulated mix for isothermal amplification of DNA and RNA targets – just add custom primers and probes • Ideal for POC diagnostic platforms or automated high-throughput instruments

PRODUCT

CAT NO. VOLUME REACTIONS

5 mL

1,000 Rxns

Air-Dryable ™ 1-Step RT-qPCR Mix

MDX095

50 mL

10,000 Rxns

0% UTM+Sputum 5% UTM+Sputum 10% UTM+Sputum

PRODUCT

CAT NO. VOLUME REACTIONS

5 mL

800 Rxns

Air-Dryable ™ RNA/DNA LAMP Mix

MDX118

50 mL

8,000 Rxns

• Concentrated 4x formulation with flexibility in final assay format (liquid or air-dried) and then can you do the same adjustments to title

High reaction specificity with little-to-no non-specific amplification Air-Dryable ™ RNA/DNA LAMP (MDX118) ( blue ) was compared to NEB WarmStart ® ( orange ) for detection of EBV DNA (1000 ipc) and MS2 RNA (1000 ipc). Reactions were incubated at 65°C for 60 min and TTR were measured at 1:10 of end fluorescence. Amplification plots of 3 technical replicates show Air-Dryable ™ RNA/DNA LAMP mix has earlier TTR and no non-specific amplification ( ) compared to NEB WarmStart ® which has detectable amplification for non-template controls (NTC) ( ).

EBV-DNA

MS2-RNA

MDX118 | NEB WarmStart ®

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RT-qPCR Master Mixes

Air-Dryable TM Direct RNA/DNA qPCR Saliva

PRODUCT HIGHLIGHTS

Sensitive detection in multiplex assays using saliva samples (up to 60% human saliva) or Universal Transport Media (up to 35% UTM) Three respiratory pathogens, Influenza A, Middle East Respiratory syndrome coronavirus (MERS-CoV) and Respiratory Syncytial Virus (RSV) were amplified in a triplex qPCR assay in the presence of 35% Universal Transport Media (UTM) with artificial sputum swab. The results illustrate that a higher performance was achieved with Air-Dryable ™ Direct RNA/DNA qPCR Saliva ( red ) compared to TaqPath RT-qPCR Mix ( black ) and UltraPlex 1-Step Tough Mix ( grey ). PRODUCT • Specimen-specific mix designed for singleplex or multiplex assays detecting RNA or DNA pathogens at very low titers from saliva or sputum samples • Ideal for POC diagnostic platforms or automated high-throughput instruments • Can be used in a wet or dry format, and compatible with a range of air-drying protocols to produce an ambient temperature stable mix CAT NO. VOLUME REACTIONS Air-Dryable ™ Direct RNA/DNA qPCR Saliva MDX131 5 mL 1,000 Rxns 50 mL 10,000 Rxns

Influenza A

MERS-CoV

RSV

Air-dried MDX131 | TaqPath RT-qPCR | UltraPlex 1-Step Tough Mix

Lyo-Ready TM Direct RNA/DNA qPCR Saliva

PRODUCT HIGHLIGHTS

• Specimen-specific mix designed for singleplex or multiplex assays detecting RNA or DNA pathogens at very low titers from saliva or sputum samples • Suitable for direct amplification from crude clinical specimens • Can be used in a wet or dry format and is compatible with a range of lyophilization protocols

PRODUCT

CAT NO. VOLUME REACTIONS

5 mL

1,000 Rxns

Lyo-Ready ™ Direct DNA qPCR Saliva

MDX132

50 mL

10,000 Rxns

5 mL

1,000 Rxns

Lyo-Ready ™ Direct RNA/DNA qPCR Saliva

MDX133

50 mL

10,000 Rxns

Sensitive detection from sputum samples containing Universal Transport Media (up to 50% UTM) Three respiratory pathogens, Influenza A, Middle East Respiratory syndrome coronavirus (MERS-CoV) and Respiratory Syncytial Virus (RSV) were amplified in a triplex qPCR assay in the presence of 50% Universal Transport Media (UTM) swab containing sputum. The results illustrate that a higher multiplexing capacity and reproducibility was achieved with lyophilized Lyo-Ready ™ Direct RNA/DNA qPCR Saliva ( red ) compared to TaqPath ™ 1-Step Multiplex Master Mix ( black ) or UltraPlex ® 1-Step ToughMix ( grey ).

Influenza A

MERS-CoV

RSV

Air-dried MDX133 | TaqPath RT-qPCR | UltraPlex 1-Step Tough Mix

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SARS-CoV-2 High Sensitivity Antibodies

For Rapid Antigen Detection Assays

Rapid antigen tests can provide a result in minutes and can be produced at a lower cost in large scales. However, the performance of a rapid antigen test is limited by the sensitivity of the antibodies used. Meridian’s monoclonal antibodies are highly sensitive for the detection of SARS-CoV-2 and they do not cross-react with seasonal coronavirus strains. They are ideal for developing reliable and sensitive rapid lateral flow antigen assays for the detection of active COVID-19 infections.

NUCLEOCAPSID ANTIBODIES

Detects omicron variant down to 1.31 x 10 2 PFU/mL and all other variants down to 1.88 x 10 2 PFU/mL in clinical samples Used in both FDA EUA and CE-marked kits Validated for lateral flow & ELISA >95% purity (Protein G) Does not cross-react with other coronaviruses, except SARS (2003)

Capture Antibody:

9548

MAb to SARS-CoV-2 NP

*pairs with both detection antibodies 9547 and 9549

Detection Antibody:

9547

MAb to SARS-CoV-2 NP

9549

MAb to SARS-CoV-2 NP

Cat# 9548/9547 detects variant strains Alpha B.1.1.7, Beta B.1.351, Gamma P.1/P.2 , Delta B.1.617.2 and Omicron B.1.1.529

TRIMERIC SPIKE ANTIBODIES

Capture Antibody:

For use in lateral flow antigen detection COVID-19 assays with a trimetric spike recombinant antigen as control (e.g: Cat# 9566) Designed to work on saliva samples (no lysis required) Novel antibody pair to the trimeric form of the Spike 1 surface glycoprotein, recognizes both the G614 and D614G strains Using a mixture of detection antibodies Cat# 9565 and #9551 in a 1:1 ratio creates a synergistic effect that increases assay sensitivity in ELISA Recognizes a linear epitope - binding is not conformation-dependent Sensitivity of approx. 300 pg/mL in ELISA Does not cross-react with SARS-CoV, HCoV-229E, HCoV-HKU1, HCoV-NL63 and HCoV-OC43

9550

MAb to SARS-CoV-2 S1 (Trimeric)

Detection Antibody:

9551

MAb to SARS-CoV-2 S1 (Trimeric)

9565

MAb to SARS-CoV-2 S1 (Trimeric)

*9551 and 9565 can be used together in a 1:1 ratio to increase assay sensitivity in ELISA

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SARS-CoV-2 Recombinant Antigens

For Serology Antibody Assays

Serologic tests are an important tool for monitoring the evolution of an outbreak. Specifically, IgG/IgM antibody tests are essential for identifying previously undiagnosed infections in the asymptomatic population. Meridian’s recombinant antigens are expressed in human mammalian cells and insect cells using proprietary expression and purification technologies. Expression in either human or insect cells provides for post-translational modifications such as glycosylation and phosphorylation which can offer significant performance advantages over E. coli expressed formats.

Wild-type SARS-CoV-2

Mutant SARS-CoV-2 Spike, S1 RBD

Trimeric Spike, S1 Protein (His-Tag) Represents amino acids 14 - 683 of SARS-CoV-2 plus the synthetic trimeric domain Spike Protein, S1 Subunit (His-Tag) Represents amino acids 14 - 683 of the SARS-CoV-2 S1 subunit Spike Protein, N-terminal Domain (NTD) S1 Subunit (His-Tag) Represents amino acids 16 - 305 of the SARS-CoV-2 N-terminal domain Spike Protein, S1 Subunit RBD (His-Tag) Represents amino acids 330 - 521 of the SARS-CoV-2 S1 subunit, RBD domain

Omicron mutant (B.1.1.529.1) Represents Omicron variant with 15 mutations on the RBD

9544

9566

L452R and T478K mutant (His-Tag) Represents B.1.617.2 (Delta Variant), a.a. 319 - 541

9606

9556

N501Y mutant (His-Tag) Represents B.1.1.7 strain, (Alpha variant) a.a. 319 - 541 K417N, E484K and N501Y mutant (His-Tag) Represents B.1.351 strain, (Beta variant) a.a. 319 - 541 K417T, E484K and N501Y mutant (His-Tag) Represents P.1 strain, (Gamma variant) a.a. 319 - 541

9567

9557

9568

9558

9569

9560

Nucleocapsid Protein (His-Tag)

L452R mutant (His-Tag) Represents Epsilon variant, a.a. 319 - 541

9598

9552

Spike Protein, S1 Subunit RBD (His-Tag)

L452R and E484Q mutant (His-Tag) Represents B.1.167 strain (Kappa variant), a.a. 319-541

9599