Fast, Reliable Protocol Delivers Distinct Benefits With Less Manpower, Expertise, and Equipment Needed – ScienceDaily

The current gold standard for COVID-19 diagnostic tests in the United States, developed by the United States Centers for Disease Control and Prevention (CDC), is PCR-based quantitative molecular tests (qPCR) that detect the presence of the viral nucleic acid. Although very accurate, these CDC-approved tests require specialized reagents, equipment, and personnel training. Additionally, multiple diagnostic kits which have been rapidly developed and introduced to the market have limitations in accuracy, cost, and delivery. Additionally, test systems currently in use are not easily adaptable to a high throughput platform to deliver the millions of tests required per day.

In view of the urgent need to develop alternative reagents and approaches to provide nucleic acid testing in the face of increased demand and potential shortages, a research team from the Schmidt College of Medicine at Florida Atlantic University has developed a Simplified COVID-19 testing protocol that offers an advantage over standard viral or universal transport medium (VTM). This test protocol can detect minimal amounts of SARS-CoV-2 using specimens collected from both upper respiratory tract swabs (nasal and throat) as well as saliva, and can be used in research laboratories with minimal equipment and expertise in molecular biology.

The protocol, published in PLOS ONE, uses TRIzol (guanidinium thiocyanate / phenol-chloroform) to purify viral RNA from different types of clinical samples, requires minimum biosafety level precautions and, given its high sensitivity, can be easily adapted to implementation strategies. in common samples. Using this simplified protocol, samples are eluted in TRIzol immediately after collection and RNA is extracted. The results showed that this protocol is as efficient, if not more, than CDC-approved silica membrane-based RNA purification microcolumns for isolating small amounts of viral and cellular RNA from several types of samples (nasal and throat swabs and saliva).

“The high sensitivity of our protocol can be useful for testing patients with low viral titers such as asymptomatic patients or testing individuals prior to quarantine. Our method also allows multiple patient samples to be pooled, reducing the number of tests required for larger populations, “said Massimo Caputi, Ph.D., senior author and professor of biomedical sciences at the Schmidt College of Medicine of the FAU. “In addition, tests can easily be performed by any research laboratory with minimal standard equipment. Since saliva can be used as a reliable source of virus, samples can be self-obtained by patients. and inactivated in TRIzol, thus eliminating the need for medical attention, personnel and higher level biosafety protocols and facilities. “

With this new approach, samples are first pooled and tested; the positive pools are then retested individually. This relatively simple solution decreases the testing resources used but results in a loss of sensitivity due to the dilution of samples from positive patients with negative samples, hence the need for very sensitive tests using biological materials, such as saliva, which can be obtained in larger quantities and can be easily preserved for retesting.

The protocol uses common chemical reagents that are in abundance and can isolate high quality RNA that can be used for several RNA sequencing assays and projects. Additionally, TRIzol samples can be stored at 4 ° C for over a week with minimal degradation and little or no loss of viral RNA. In addition, the possibility of using saliva samples, which are as sensitive or more reliable than nasopharyngeal swabs, offers an attractive alternative to samples. Nose and throat swabs are the most common upper respiratory tract samples used for diagnostic testing for COVID-19. However, collecting these types of specimens can cause discomfort, bleeding, and require close contact between health workers and patients, posing a risk of transmission.

In the most commonly used COVID-19 testing protocols, a healthcare professional takes a nasal or throat swab and transfers it to a vial containing a few milliliters of VTM. The sample is then transported to a laboratory for analysis. Transportation and storage can take anywhere from a few hours to a few days depending on the distance and processing times from the nearest clinical laboratory. The CDC recommends that samples be stored at 2-8 ° C for up to 72 hours after collection and at -70 ° C or below for longer periods. However, the logistics of having multiple sample collection points, blockages in the reagent supply chain and sharp increases in test demand due to local outbreaks can generate unexpected delays in sample processing. .

“We can expect strong demand for COVID-19 testing for the foreseeable future as testing of the general population and asymptomatic individuals becomes more widespread,” said Janet Robishaw, Ph.D., co-author, associate dean Principal for Research and Chairman of the Department of Biomedical Science at the Schmidt College of Medicine at FAU. “The lack of control of the pandemic in many underdeveloped countries as well as the continued escalation of COVID-19 in the United States are also compelling reasons to step up testing efforts. We hope that a combination of testing approaches, including protocols like ours, may be the most effective way to address current and future testing gaps. “

The co-authors of the study are Sean Paz; Christopher Mauer; and Anastasia Richtie; graduate students from FAU’s Schmidt College of Medicine.

This work was supported by the Florida Blue Foundation grant, “Development of Predictive Algorithms for COVID-19 Infection in FAU Healthcare Workers.”

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Material provided by Florida Atlantic University. Original written by Gisele Galoustian. Note: Content can be changed for style and length.