SARS-CoV-2 saliva testing that omits the RNA extraction step

By Dr. Liji Thomas, MDJun 21 2020

Even as the COVID-19 pandemic progresses throughout the world, the lack of consistent diagnostic testing has been a bugbear, preventing public health control of the spread of infection. A new study by researchers at the University of Illinois Urbana-Champaign and published on the preprint server bioRxiv* in June 2020 reports a new testing method that uses saliva, does not require RNA extraction, and can be scaled up quickly and inexpensively.

The researchers describe their findings thus, “This saliva-based process is operationally simple, utilizes readily available materials, and can be easily implemented by existing testing sites, thus allowing for high-throughput, rapid, and repeat testing of large populations.”

Novel Coronavirus SARS-CoV-2 Transmission electron micrograph of a SARS-CoV-2 virus particle, isolated from a patient. Image captured and color-enhanced at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

The Need for Simple Rapid Scalable Testing

The study was motivated by the need for “convenient, repeatable, large-scale molecular testing for SARS-CoV-2,” which “would be a key weapon to help control the COVID-19 pandemic.” At present, nasopharyngeal (NP), nasal and oropharyngeal swabs are used for routine PCR testing. These are not only invasive but need many bits of equipment that are not readily available, making repeat testing a challenging task. These include the need for swabs, personal protective equipment (PPE), viral transport media (VTM), and kits for RNA isolation and purification.

Secondly, the risk posed by such testing is significant as the virus is spread primarily through aerosols and respiratory droplets. However, saliva is an excellent alternative sample source. The current study aims to understand the advantages of using saliva for SARS-CoV-2 testing.

The need for such a study lies in the possibility it offers of a cost-effective, high-throughput platform for testing thousands of individuals, if need be, in a single day. This could allow the repeated testing of individuals to identify groups of people who can safely interact to continue business operations, for instance, subject to their isolating from other possible carriers or patients.

The Current Study: Direct PCR from Saliva

Several earlier reports have shown that direct detection of the virus from swabs or VTM without RNA extraction steps. The current study examines direct detection potential from saliva, to set up a simple, rapid, and inexpensive workflow capable of being used for any testing laboratory by RT-PCR.

The researchers compared NP swabs with saliva specimens, because saliva is easy to collect, is known to contain the virus, is a potential source of viral transmission, and can be used to develop a workflow that can test up to 10,000 people a day. Not much information is available on the way the viral load changes over time in saliva, but what is known suggests that it is highest in the first week of infection, that is, in the incubation period.

On the other hand, saliva no longer contains live virus capable of being cultured after 10 days from the earliest symptom, unlike NP swabs, which are still positive in the convalescent stage. However, the patient is non-infectious by then.

Inactivation, Buffering and Additives

The researchers used a couple of versions of the inactivated virus via gamma-irradiation and heating, respectively. They found that following heating at 95°C for 30 minutes, they achieved 100% identification of all SARS-CoV-2 in all samples. A short heating time of 5 minutes does not provide the same sensitivity because salivary inhibitors of RT-PCR are not inactivated.

In short, the study says, “proper heating of patient samples allows for virus detection without the need for RNA extraction, with the added benefit of inactivating the samples, thus substantially reducing biohazard risks.”

They also found that using TBE buffer with the saliva samples led to outstanding detection rates for RNA without RNA extraction. Sometimes detergent is added to improve saliva viscosity and improve detection rates. When the current study evaluated the effects of adding either or both TBE and the detergent Tween 20 before or after heating, they concluded: “the safest and most streamlined protocol would be: collection of saliva samples, heat at 95^oC for 30 min, add TBE buffer and Tween 20, followed by RT-qPCR.”

The virus is shed into saliva over an extensive range from 10,000 to 10 billion copies per mL. The current limit of detection (LOD) was determined to be about 1,000 copies/mL after RNA extraction, and in saliva, about 5,600 copies/mL. With the current process, the LOD was determined to be about 500 copies/mL for an irradiated virus, and about 5,000 copies/mL for heat-inactivated virus. Equivalent results were obtained with multiple analytical tools.

The researchers then examined the suitability of the sample for storage. They found that saliva can be stored in stable form under different storage conditions, can be heated in vessels of varying volume but that centrifugation affects direct RT-PCR results adversely.

Initial Clinical Assessment Shows Excellent Results

Using clinical specimens, they found that with duplicate testing of samples, the direct detection of salivary virus by RT-PCR had 100% sensitivity, specificity, positive predictive value, and negative predictive value. Still, false negative and false positive was 0. These preliminary assessments were found to be very promising and should prompt further direct comparisons of this method with NP-swab detection, using the optimized workflow.

The advantages of this method are many. Not only is it simple, it saves time and effort in obtaining clinical specimens. By not doing RNA extraction and purification, the use of reagents and other laboratory equipment is reduced, which leads to a saving of about $10 per test, and more if pooled samples are used. Its compatibility with many platforms reduces the chances of supply bottlenecks.

The researchers sum up: “Described herein is a sensitive diagnostic method for SARS-CoV-2 that is operationally simple, bypasses supply chain bottlenecks, evaluates a clinically relevant infectious fluid, is appropriate for large scale repeat testing, is cost-effective, and can be readily adopted by other laboratories. Large scale SARS-CoV-2 testing will be a powerful weapon in preventing the spread of this virus and helping to control the COVID-19 pandemic.”

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.