Improved Sensitivity and Multiplexing in COVID-19 Diagnostic Assays

The key to containing a pandemic is large-scale rapid diagnostic testing. Although the gold standard for viral diagnostics is nucleic acid testing, there are other types of equally important tests such as serological and antigen tests. Each of these play distinct roles in diagnostics at the hospital, point-of-care, and population levels.

An initial problem with trying to quickly implement nucleic acid testing during the early stages of the COVID-19 pandemic was that it required, “sophisticated equipment, highly trained personnel, and the results were usually generated with a duration of days,” Yuning Chen, Research and Development Manager at Sino Biological, explains.

Recent advances in instrumentation and reagents have addressed these challenges amid emerging variants. In addition, new diagnostic tests are more sensitive and able to detect low amounts of viral load, which is key for controlling spread because around 60% of COVID-19 transmissions have been from asymptomatic carriers. The development of multiplexed diagnostics allows clinicians to detect multiple respiratory viruses at once that may not be distinguishable by symptoms alone, further increasing the efficiency of diagnostic assays. These technologies are not necessarily specific for the COVID-19 pandemic. As Nicholas Abuid, Field Application Scientist at ACROBiosystems, explains, “The current innovations being developed in the diagnostics field can be applied for several other diseases such as cardiovascular, neurological, and cancer, which could be cured with early diagnosis.”

Instrumentation

At the beginning of the COVID-19 pandemic, fluorescent qPCR was used extensively. Two problems that limited the efficiency and global use include detection time and stability associated with long-distance transportation of reagents. “If room temperature transportation is used, the enzyme activity in liquid reagents will be affected, which will result in a decrease in detection sensitivity and accuracy,” As Elio Yan, CTO of Bioteke Corporation, explains.

To address these problems, Bioteke developed a new generation of ultra-fast fluorescent qPCR instruments (BTK-8) and is utilizing freeze-drying technology. Yan notes that the new instruments have “excellent performance that is reflected in the temperature change rate of up to 12°C/s, which can complete eight PCR reactions within 30 minutes.” In addition, Bioteke “screened different reverse transcriptases and hot-start enzymes, tested different reaction systems, and combined them with the advantages of freeze-drying technology,” allowing the reagents to be transported at room temperature for one month without affecting the sensitivity and accuracy. These advances have increased accessibility to rapid diagnostics around the world.

Nucleic acid tests

Nucleic acid tests use PCR technology to detect the genetic material of SARS-CoV-2. The CDC initially submitted an Emergency Use Authorization (EUA) for the 2019-nCoV Real Time RT-PCR Diagnostic Panel in February 2020 to address the need for COVID-19 diagnostics. This panel will be discontinued after 2021 because of the authorization of high-throughput alternatives and multiplexed assays.

One such alternative is the PKamp™ Respiratory SARS-CoV-2 RT-PCR Panel Assay, which reportedly has the lowest limit of detection (LoD) among the authorized COVID-19 molecular diagnostic tests. This assay targets two specific genomic regions of SARS-CoV-2: the nucleocapsid (N) and the ORF1ab genes. Importantly, mutations in the spike protein do not affect the safety and effectiveness of the assay. The increased sensitivity of the assay also allows samples to be pooled together for batch testing.

Arvind Kothandaraman, Managing Director of Specialty Diagnostics at Revvity, explains that this batch testing “processes samples efficiently without increasing resources. If a pooled test is negative, all the individual samples are considered negative. If the pooled test is positive, each of the individual samples in that pool should be tested again separately to determine which of the samples are positive.” Along with automation, this assay helps alleviate test shortages. Another key feature of this assay is the ability to perform multiplexed syndromic testing of respiratory viruses, namely, SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV), that cause similar symptoms in patients and thus may not be distinguishable based on symptoms alone.

Reagents

In addition to nucleic acid tests, serology tests such as antibody and antigen tests are an effective diagnostic tool. The key ingredients for developing serology tests are high-quality recombinant proteins and antibodies. As Chen explains, “Recombinant proteins are produced in suitable host cells to mimic their naturally occurring counterparts, which enables the detection of serum antibodies against those proteins and in the meantime, generation of highly sensitive and specific antibodies to detect the presence of virus fragments in the serum.” Amid the pandemic, high-throughput protein expression and antibody screening platforms have been developed to generate variant proteins, allowing researchers to assess the impact of mutations on diagnostic assays. Chen continues, “The technologies established during the COVID response can also be applied to other infectious diseases such as influenza, RSV, HCV, etc. for the development of rapid and accurate serology tests.”

Variants and beyond

Over the past year and a half, numerous SARS-CoV-2 variants have arisen with varying degrees of transmissibility and symptom severity. To keep up with new mutations, ACROBiosystems continues to produce reagents that satisfy the needs of diagnostic research and the emergence of new variants. Abuid explains that they are “rapidly updating the new mutations present in each of these variants, including the delta plus, lambda, and mu.” Each of these variants has different mutations in the different components, and ACROBiosystems will continue launching proteins that can help researchers using their reagents develop new diagnostics.

Kothandaraman believes that COVID-19 is here to stay and will become an endemic disease. Therefore, keeping track of new variants is key to managing outbreaks. “Tracking the spread and evolution of SARS-CoV-2 using next-generation sequencing and other methods is imperative for the adaptation of COVID-19 diagnostics, vaccines, therapeutics as well as future pandemic preparedness,” he adds.