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We have so far discussed the most current diagnostic tests that have been authorized by the Food and Drugs Administration (FDA) i.e Cepheid’s Xpert Xpress SARS-CoV-2 Test and Abbott’s ID NOW COVID-19 Test. They both qualify for the rapid test with each giving results within 45 minutes and 5-13 minutes respectively. They apply different methodologies but they both aim at detecting the SARS-CoV-2 RNA nucleic acids from a nasal swab or nasal wash samples.
Earlier before these two were designed, on March, 18th, 2020, Abbott had developed the Real-Time SARS-CoV-2 Assay. This is an automated Assay that runs on Abbott’s m2000 RealTime system aiming at detecting SARS-CoV-2 RNA by use of Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).
However, this diagnostic test is to be used only by laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests.
Objectives of Abbott RealTime SARS-CoV-2 assay
- For qualitative detection of nucleic acid from the SARS-CoV-2 in nasopharyngeal (NP) and oropharyngeal (OP) swabs from patients suspected of COVID-19.
- For the detection of COVID-19 in patients suspected of COVID-19 by their healthcare provider.
Figure: Abbott RealTime SARS-CoV-2 assay. Image Source: Abbott Laboratories
Principle of Abbott RealTime SARS-CoV-2 assay
This test has bee designed to identify the virus by targeting small amounts of the viral RNA and amplify portions of the genome until there is enough for detection. It is a fully automated diagnostic test that has been engineered with:
- Dual target assay for RdRp and N-genes
- Qualitative detection of nucleic acids from SARS-CoV-2
- maxRatio data analysis removes operator subjectivity
The Abbott Real-Time SARS-CoV-2 Assay test is a real-time reverse transcription-polymerase chain reaction (qRT-PCR) done on the Abbott m2000 System. The primer and the test probe are designed to detect SARS-CoV-2 RNA from a specimen collected from suspected cases of COVID-19. These specimens are collected from the nasal, nasopharyngeal and oropharyngeal.
The Abbott Real-Time SARS-COV-2 Assay uses two reagents:
- Abbott RealTime SARS-CoV-2 Amplification Reagent Kit
- Abbott RealTime SARS-CoV-2 Control Kit
The Abbott RealTimeSARS-CoV-2 assay is a dual target assay for the RdRp and N genes. An RNA sequence that is unrelated to the SARS-CoV-2 target sequence is introduced into each specimen at the beginning of sample preparation and amplified by RT-PCR. This serves as the internal control for the Test i.e it ensures that the process has run correctly for each sample. The Abbott RealTime SARS-CoV-2 assay detects the SARS-CoV-2 virus and IC target sequences through the use of target-specific fluorescent-labeled oligonucleotide probes. The probes do not generate a signal unless they are specifically bound to the amplified product. The two SARS-CoV-2-specific probes are labeled with the same fluorophore and the IC-specific probe is labeled with a different fluorophore, thus allowing for simultaneous detection of both SARS-CoV-2 and IC amplified products in the same reaction well.
The Abbott RealTime SARS-CoV-2 assay is performed on the Abbott m2000 System consisting of a sample preparation unit, the Abbott m2000sp, and an amplification and detection unit, the Abbott m2000rt. Application parameters specific to the Abbott RealTime SARS-CoV-2 assay are contained on an assay-specific application specification file, distributed electronically, stored on portable media and loaded onto the Abbott m2000sp and Abbott m2000rt instruments.
Procedure for Abbott RealTime SARS-CoV-2 assay
The Abbott Real-Time SARS-CoV-2 assay amplification reagents are:
- Thermostable rTth PolymeraseEnzyme
Preparation for the Sample
The Abbott m2000sp uses an automated sample preparation protocol by use of a magnetic micro-particle based protocol and reagents, known as the Abbott mSample Preparation System DNA. It processes 0.5ml of the specimen from the nasal, nasopharyngeal and oropharyngeal swabs.
Initial, the internal controls (positive and negative controls) are processed as you start the sample preparation, for each test order to evaluate the run validity. During the sample preparation procedure, SARS-CoV-2 Virions are disrupted by guanidine isothiocyanate, capturing nucleic acids on the magnetic particles and the inhibitors and unbound specimens are removed by a washing step. The bound nucleic acids are washed off the microparticles with a buffer and transferred to a 96 deep-well plate, and they are ready for amplification. The Internal Control (IC) is introduced into each specimen at the beginning of the sample preparation process to demonstrate that the process was completed correctly for each specimen and control.
The sample preparation procedure is important to extract and concentrate the target nucleic acids (RNA and DNA) making the target accessible for amplification and removal of potential inhibitors of amplification form the extract.
Generally, the Abbott m2000sp automated instrument is used to prepare the sample for the Abbott real-Time SARS-CoV-2 assay, providing an automated specimen transferred and assembled in the Abbott 96-deep well Optical reaction Plate.
Reaction Plate Assembly
The Abbott m2000sp combines the Abbott Real-Time SARS-CoV-2assay amplification reagent components (SARS-CoV 2 Oligonucleotide Reagent, Thermostable rTth Polymerase Enzyme, and Activation Reagent). The Abbott m2000sp dispenses the resulting master mix to the Abbott 96-Well Optical Reaction Plate along with aliquots of the nucleic acid samples prepared by the Abbott m2000sp. The plate is ready, after manual application of the optical seal, for transfer to the Abbott m2000rt.
- During the amplification reaction on the Abbott m2000rt, the target RNA is converted to cDNA by the reverse transcriptase activity of the thermostable rTth DNA polymerase.
- Annealation of the SARS-CoV-2 and IC reverse primers to their targets and they extend during the prolonged incubation period.
- Denaturation by raising the temperature of the reaction above the melting point of the double-stranded cDNA (an RNA product).
- A second primer then anneals to the cDNA strand and it is extended by DNA polymerase activity of the rTth enzyme creating a double-stranded DNA product.
During each round of thermal cycling, amplification products dissociate to single strands at a high temperature allowing primer annealing and extension as the temperature is lowered. Exponential amplification of the product is achieved through repeated cycling between high and low temperatures, resulting in a billion-fold or greater amplification of target sequences. The amplification of the three targets (SARS-CoV-2 RdRp, SARS-CoV-2 N, and IC) takes place simultaneously in the same reaction. The target sequences for the Abbott Real-Time SARS-CoV-2 assay are in the SARS-CoV-2 RdRp and N genes of the SARS-CoV-2 genome. The selected target sequences are highly conserved and also specific to this strain of coronavirus.
The IC target sequence is derived from the hydroxypyruvate reductase gene from the pumpkin plant, Cucurbita pepo, and is delivered in an Armored RNA® particle that has been diluted in negative human plasma.
During the read cycles of amplification on the Abbott m2000rt, the temperature is lowered further to allow fluorescent detection of amplification products as the SARS-CoV-2 and IC probes anneal to their targets (real-time fluorescence detection). The SARS-CoV-2 probes have a fluorescent moiety that is covalently linked to the 5′end and has a quencher molecule at its 3′end. In the absence of target sequences, the probes adopt a conformation that brings the quencher close enough to the excited fluorophore to absorb its energy before it can be fluorescently emitted. When the probe binds to its complementary sequence in the target, the fluorophore and the quencher are held apart, allowing fluorescent emission and detection. The IC probe is a single-stranded DNA oligonucleotide with a fluorophore at the 5′ end and a quencher at the 3′ end. In the absence of IC target sequences, probe fluorescence is quenched. In the presence of IC target sequences, probe hybridization to complementary sequences separates the fluorophore and the quencher and allows fluorescent emission and detection. The SARS-CoV-2 and IC-specific probes are each labeled with a different fluorophore, thus allowing for simultaneous detection of both amplified products.
Results and interpretation of Abbott RealTime SARS-CoV-2 assay
A positive test result indicates that RNA from SARS-CoV-2 was detected and the patient has COVID-19. Therefore, it is also likely that you may be placed in isolation to avoid spreading the virus to others. Correlate the positive results with the patient’s clinical diagnosis and epidemiological data when making the final diagnosis and patient management decision according to the CDC guidelines.
A negative test result means the RNA for SARS-CoV-2 was absent in the specimen above the limit of detection. Note that a negative result does not rule out COVID-19, consider false-negative results as well and do a followup on the patient’s clinical diagnosis, epidemiological data and ensure other tests for other illnesses are negative as well.
Advantages of Abbott RealTime SARS-CoV-2 assay
- It can test many specimens within a short time, in 100s.
- There is less likelihood of false-negative and false-positive results.
Disadvantages of Abbott RealTime SARS-CoV-2 assay
- So far it can only be used in the US as of now since it has only been authorized to be used there.
- It can only be done on an m2000 Real-Time platform of the Abbott Molecular Company.
References and Sources
- FDA Emergency Use Authorisation: Abbott Real-Time SARS-CoV-2 Assay
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