A major challenge concerning the COVID-19 virus is its detection. While the general public is frustrated at the length of time required for COVID-19 testing or developing new test methods, those in the scientific community understand that this is due to the construction of viruses in general, and the logistics of testing thousands of COVID-19 specimens.
Before we can treat new viruses or gauge their widespread impact on the general population, we must detect them. So, how do we confirm COVID-19 viral infections?
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
RT-PCR is one of the most accurate, sensitive, and widely used technologies for COVID-19 clinical diagnostic testing. Viruses have genetic material, either two-strand DNA or single-strand RNA. The genome of SARS-CoV-2 has single-stranded RNA. For SARS-CoV-2 diagnostic tests, RT-PCR is used to convert the viral RNA to cDNA, which is subsequently amplified to detect the presence of the virus in COVID-19 specimens.
The two methods used for RT-PCR are one-step and two-step RT-PCR.
One-step PCR is ideally suited for high-throughput screening. The reverse transcriptase step is performed in a single tube—the same tube the PCR reaction occurs. While preparation is simple and rapid, it does have the drawback of being generally less sensitive as no ability exists to tweak the reactions separately as they occur in the same tube. However, a distinct advantage is the one-step process works well with robotic liquid handling systems and is easier to configure for automated analysis.
The second method involves the step of producing complementary DNA, or cDNA. This cDNA is then added to the PCR reaction in a different tube, hence the second step. While preparation and analysis time is greater, it is highly sensitive and ideal when the reaction is performed with a limiting amount of starting material. Two-step RT-PCR does require additional pipetting steps, thus adding additional points of error and potential contamination to the process. The two-step process is more difficult to automate, so high-throughput analyses are hindered.
Quantitative PCR, or qPCR, incorporates fluorescence, either as intercalating dyes or sequence-specific probes into nascent DNA.
This is important because it allows the use of a digital capture in real time as opposed to the conventional gel electrophoresis method.
A misused term in some circles, high throughput can mean analyzing multiple samples at once, or performing multiple analyses on one sample. For COVID-19 work, we are concerned with the former.
Thermocyclers in which PCR is executed have evolved since the days of 96 well plate limitations. Now, 384 well plates are common, and we are now seeing well plates capable of handling 1536, or even, in the case of Wafergen’s SmartChip Real-Time PCR, 5,184 wells. The more wells, the more samples, and thus the higher the throughput.
We all produce antibodies from specialized white blood cells called B lymphocytes, or B cells. When an alien substance, an antigen, enters our bodies, a sophisticated mechanism kicks in. Those B cells clone and secrete millions of antibodies into our bloodstream and lymphatic system. This is often a very routine and reliable defense against viruses, which is why we simply don’t perish from every viral infection we acquire.
While RT-PCR focuses on the direct detection of an antigen, there is another COVID-19 diagnostic test that involves the search for the antibodies reacting to the virus. Unlike RT-PCR testing, which is performed on swabs of the nose or throat, antibody detection is a serological test performed on blood samples.
Called an immunoassay, the detection of the antibodies produced in response to a COVID-19 infection are, for now, marginally useful because they are not reliable as the sole diagnostic tool to confirm COVID-19 infection, rather, they confirm that the body has been exposed to COVID-19 at some point.
Antibody detection is a fantastic tool for epidemiological studies, meaning tracking the spread of the virus in the population, but not the ideal diagnostic tool. What the presence of COVID-19 antibodies does tell us is that the person has developed COVID-19 defenses and is likely immune to further infections. This could be a key tool to know who is at risk when working with patients suspected of having been infected with COVID-19.
Enzyme-linked Immunosorbent Assay (ELISA)
Antibody detection takes several forms. The venerable ELISA was first described in 1971. While ELISA is a relatively simple procedure and very good at relating antigens to antibodies, it is labor intensive and carries with it the significant cost of culture media and a high possibility of false positive/negative readings. A plus for ELISA analyses is the time required is only in the range of one to five hours. Still, ELISA testing is highly automated and has been interfaced to commercial LIMS for decades.
This antibody test relies upon examining patient antibodies and their ability to prevent an infection in a laboratory setting. Also a serological test requiring blood or serum samples, this assay is regarded for its ability to detect the threshold of antibodies required to block virus replication. Why is this important? For starters, we do not have enough COVID-19 data to know if the antibodies produced to combat the infection will result in protective immunity, and if it does, is it long-lasting immunity?
Having said that, neutralization assays are not expedient, requiring three to five days to execute. Regardless, antibody detection remains a paramount focus.
Upcoming tests based on DNA sequencing
One of the upcoming tests for COVID-19 diagnostic testing is based on Sanger Sequencing, a DNA sequencing method. The test is based on the sequencing of a single amplicon from the viral genome and analysis of the sequencing data using computational methods. The Sanger sequencing-based COVID-19 testing method can be used by any clinical laboratory once approved by the US Food and Drug Administration (FDA). This method has the potential to increase the COVID-19 testing capacity by several folds due to the high-throughput of Sanger sequencing instruments. Some companies are also developing NGS-based tests for the diagnostic testing of COVID-19 specimens. The test is based on the sequencing of the entire viral genome to create hundreds of targets for the detection of coronavirus in COVID-19 specimens. The test is not only expected to help in diagnostic testing, but also in the characterization of the viral genome, thereby accelerating viral mutation studies and the development of vaccines. Once these tests are approved by the FDA, they can be used to expedite the testing of COVID-19 specimens.
Laboratory information management systems (LIMS) and the information avalanche
With so much attention being paid to the diagnosis and treatment of COVID-19 victims, how are clinicians and researchers managing the incredible overload of information?
High-volume coronavirus diagnostic testing requires the relay of instrumental results to a commercial LIMS. There was a time when most instrumental results were merely transcribed from paper instrument printouts or computer screens to a LIMS, but with the advent of digital technology, instrument interfacing to a LIMS has all but eliminated manual transcription.
Interfacing can be either unidirectional or bidirectional. Unidirectional interfaces usually rely upon instruments pushing output files to shared directories that are monitored by the LIMS that detect the presence of new data files which are then parsed and uploaded into the LIMS. Bi-directional interfaces may take the form of instrument systems that have direct Application Programming Interfaces (API) to the LIMS allowing for seamless communication between the systems.
Whether a LIMS has an onboard instrument interfacing tool, or is reliant upon third-party middleware, the rapid and reliable transfer of instrumental data to a LIMS is both rapid, highly-reliable, and error free.
LIMS have flexible analytic and reporting tools ranging from bespoke querying functionality for one-off data analysis needs to reporting tools that can be easily configured by non-IT personnel.
Reporting tools can be used to provide a little or a lot of detailed formatting as might be required by regulatory agencies.
An in the cloud LIMS requires no host computer, no in-house maintenance, and no worries that the most current version of the software is in play. Analyzing laboratories can concentrate on the Herculean task of processing many thousands of samples, while ensuring that the critical tracking of results and attribution of those results back to the patients is well in hand.
We are winning many battles against the COVID-19 virus, but to win the war, we must combine accurate diagnostic tools with COVID-19 lab software solutions so that we may quickly detect COVID-19 infections across millions of patients, automate result reporting, and minimize turnaround time. Until that unified attack plan is implemented, we are all at a disadvantage, but the good news is we will ultimately win the war.
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