New testing protocols for SARS-CoV-2 can handle a huge number of tests inside 48 hours

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• Source: IMBA- Institute of Molecular Biotechnology of the Austrian Academy of Sciences


• Date: 25 May,2021

Researchers in the Vienna BioCenter designed a testing protocol for SARS-CoV-2 that can process tens of thousands of samples in less than 48 hours. The method, called SARSeq, is printed in the journal Nature Communications and might be adapted to many more pathogens.

The COVID-19 pandemic has lasted more than a year and continues to impact our lives tremendously. Even though some countries have launched rapid vaccination attempts, many still await large-scale immunization strategies and effective antiviral therapies – before that happens, the world desperately needs to regain a semblance of normalcy.

1 way to bring us closer to that point is massive parallel testing. Molecular tests that detect the presence of SARS-CoV-2 have been the best way to isolate positive cases and contain the spread of this virus. Several methods have come forward, some that detect viral proteins from nasopharyngeal swabs (for instance, antigen evaluations ), and some that detect the presence of viral RNA from swabs, gargle samples, or saliva samples (such as reverse transcription and polymerase chain reaction tests, or RT-PCR).

Although antigen tests facilitate some logistical aspects of mass testing, their detection power is relatively weak – infected people carrying low amounts of virus remain unnoticed and may continue to infect other men and women. PCR tests, on the other hand, are more sensitive because they multiply fragments of the viral genome prior to scanning samples for the virus. However, they rely on the detection of fluorescent labels that label viral sequences, meaning pooling samples coming from other individuals makes the process rather inefficient: if a pool tests positive, all of the samples inside the pool must be analyzed again separately to identify the source of the fluorescent signal. Too many machines needed, too costly, too slow.

During the very first lockdown, scientists in the Vienna BioCenter were mulling over the situation: there had to be a way to scale up testing. Ulrich Elling, group leader at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), and Luisa Cochella, group leader in the Research Institute of Molecular Pathology (IMP), decided to channel their frustration into an innovative solution. IMP group leader Alexander Stark and IMBA postdoc Ramesh Yelangandula joined their efforts, and the project took off.

Amplifying the viral material from individual samples to a maximum homogenizes its quantity across positive samples, making SARSeq highly sensitive,. Within the thousands of samples that we could test simultaneously, some may contain up to 10 million times more coronavirus particles than others – if we pooled such samples before amplification, those with high amounts of viral material could mask other positive cases.”

Luisa Cochella, Group Leader, Research Institute of Molecular Pathology, Institute of Molecular Biotechnology of the Australian Academy of Sciences

Combining their expertise in genomics, RNA biochemistry and information analysis, they developed a method that could enable large groups to be analyzed for SARS-CoV-2 with similar sensitivity as routine PCR tests. SARSeq, or’Saliva Analysis by RNA sequencing’, achieves high sensitivity, specificity, and the capability to process up to 36,000 samples in less than two days. The method is currently published in the journal Nature Communications.

Then, a fragment of viral RNA unique to SARS-CoV-2 – the nucleocapsid gene – is converted to DNA and PCR-amplified in any well that contains it.

What distinguishes this first step to the typical PCR test is that each sample receives a unique pair of short DNA sequences – or barcodes – that attach to the amplifying viral DNA. In another amplification step, all of the samples from 1 plate are pooled into a single well, which receives another set of unique DNA barcodes.

The contents of numerous plates can be pooled once more, as the DNA molecules from each sample carry a unique combination of 2 sets of barcodes. This pooling and barcoding strategy makes SARSeq highly scalable and specific.

The NGS machine processes the pooled samples and tells us which samples contained any SARS-CoV-2 material. The barcodes enable us to distinguish each positive sample from the others, and trace it back to a patient,” says Ramesh Yelagandula, first author of the study. Moreover, the NGS-based method allows to test several RNAs in parallel, such as RNAs that control the sample quality or RNAs from other pathogens for differential diagnostics.

“The Next Generation Sequencing facility along with other colleagues in the Vienna BioCenter were of enormous help to develop and optimize the method,” says Alexander Stark. “With our machines, home-made enzymes, and analysis pipeline, we expect each test to cost less than five Euro.”

The testing process can operate in parallel to existing diagnostics, while being independent of the bottlenecks in supply chains. Therefore, it does not compete with other testing methods for reagents or gear.

“We developed SARSeq to try and circumvent the limitations of different tests, and to process thousands of samples in parallel. Not only is it an exceptional technique to detect SARS-CoV-2, but it can also be applied to other respiratory pathogens like the flu virus, the common cold rhinoviruses, and possibly many others,” says Ulrich Elling.

The principles behind SARSeq are simple and adaptable to any respiratory pathogen.