Fast, reliable protocol offers distinct advantages with less work, expertise and necessary equipment – ScienceDaily

To properly monitor and help curb the spread of COVID-19, only a few million diagnostic tests are needed daily in the United States alone. There is still widespread testing for COVID-19 in the U.S., and many clinical diagnostic protocols require extensive human labor and materials that could address supply shortages and raise biosecurity concerns.

The current gold standard for the COVID-19 diagnostic test in the United States, developed by the U.S. Centers for Disease Control and Prevention (CDC), is quantitative PCR-based molecular tests (qPCRs) that detect the presence of viral nucleic acid. Although very accurate, these tests approved by the CDC require specialized reagents, equipment, and staff training. In addition, several diagnostic kits that are rapidly developed and introduced to the market have limitations on accuracy, cost, and distribution. Moreover, the test systems currently in use are not easily adaptable to the high-bandwidth platform to deliver the required millions of tests per day.

Given 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 Schwidt School of Medicine at the University of Florida Atlantic has developed a simplified COVID-19 test protocol that offers special advantage over on standard viral or universal transport medium (VTM). This test protocol can detect minimal amounts of SARS-CoV-2 using samples of both upper respiratory tract swabs (nose and throat) as well as saliva, and can be used in research laboratories with minimal equipment and expertise in molecular biology.

Protocol published in PLOS ONEuses TRIzol (guanidinium thiocyanate / phenol-chloroform) to purify viral RNA from different types of clinical specimens, requires minimal biosecurity precautions and, given its high sensitivity, can be easily adapted to a sample aggregation strategy. Using this simplified protocol, samples were eluted into TRIzole immediately after collection and RNA extraction. The results showed that this protocol was equally effective, if not larger than the silicon membrane-based RNA purification microcolumns, in isolating small amounts of viral and cellular RNA from multiple types of samples (nasal and throat and saliva swabs).

“The high sensitivity of our protocol can be useful in testing patients with low viral titers, such as asymptomatic patients, or in 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 Dr. Massimo Caputi, lead author and professor of biomedical sciences at FAU’s Schmidt School of Medicine. “In addition, the test can be easily conducted by any research laboratory equipped with a minimum of standard equipment. Because saliva can be used as a reliable source of the virus, patients can obtain samples themselves and inactivate them in TRIzole, eliminating the need for medical personnel and protocols and biosafety facilities at a higher level. “

With this new approach, samples are first pooled and tested; the positive pools are then retested individually. This relatively simple solution reduces the resources used for testing, but results in loss of sensitivity by diluting positive patient samples with negative ones, hence the need for highly sensitive tests using biological materials, such as saliva, which can be obtained in larger quantities and can be easily preserved for re- testing.

The protocol uses common chemical reagents that are abundant and can isolate high-quality RNA that can be used for multiple RNA sequencing tests and projects. In addition, samples in TRIzole can be stored at 4 C for more than a week with minimal degradation and little or no loss of viral RNA. Moreover, the ability to use saliva samples that are more sensitive or reliable than nasopharyngeal swabs offers an attractive alternative to the sample. Nasal and throat swabs are the most common upper airway specimens used for the COVID-19 diagnostic test. However, collecting these types of samples can cause discomfort, bleeding, and requires close contact between healthcare professionals and patients, which poses a risk of transmission.

In the most commonly used COVID-19 test protocols, a healthcare professional takes a swab of the nose or throat and transfers it to a bottle containing a few milliliters of VTM. The sample is then transported to the laboratory for testing. Transport and storage can take from a few hours to a few days, depending on the distance and processing time of the nearest clinical laboratory. The CDC recommends that samples be stored at 2 to 8 ° C for up to 72 hours after collection and at -70 ° C or lower for an extended period of time. However, the logistics of having multiple sampling points, reagent supply chain downtime, and a sudden increase in test demand due to local outbreaks can generate unexpected delays in sample processing.

“We can expect high demand for testing on COVID-19 in the foreseeable future as testing of the general population and asymptomatic individuals expands,” said Dr. Janet Robishaw, co-author, senior research associate and chair of the Department of Biomedical Sciences at FAU’s Schmidt School of Medicine. “The lack of pandemic control in many underdeveloped countries, as well as the continued escalation of COVID-19 in the US, are also necessary reasons to increase testing efforts. We hope that a combination of testing approaches, including protocols like ours, will be the most effective way to fill current and future testing gaps. “

The co-authors of the study are Sean Paz; Christopher Mauer; and Anastasia Richtie; graduates at FAU Schmidt School of Medicine.

This work was supported by a grant from the Florida Blue Foundation, “Development of predictive algorithms for COVID-19 infection in FAU health workers.”

Source of the story:

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

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