Diagnosis of any infectious disease is vital for opportune treatment and to prevent dissemination. RT-qPCR tests for detection of SARS-CoV-2, the causative agent for COVID-19, are ideal in a hospital environment. However, mass testing requires cheaper and simpler tests, especially in settings that lack sophisticated machinery. The most common current diagnostic method is based on nasopharyngeal sample collection, RNA extraction, and RT-qPCR for amplification and detection of viral nucleic acids.
Here, we show that samples obtained from nasopharyngeal swabs in VTM and in saliva can be used with or without RNA purification in an isothermal loop-mediated amplification (LAMP)-based assay, with 60–93% sensitivity for SARS-CoV-2 detection as compared to standard RT-qPCR tests. A series of simple modifications to standard RT-LAMP published methods to stabilize pH fluctuations due to salivary acidity resulted in a significant improvement in reliability, opening new avenues for efficient, low-cost testing of COVID-19 infection.
The years 2019 to 2021 will be remembered for the coronavirus-disease 2019 (COVID-19) pandemic. The disease toll in the world has surpassed 142.5 million cases and more than 3 million deaths and does not seem to have decreased its rate of contagion in the past months. In the US alone more than 31.7 million cases and over five hundred and sixty eighty thousand deaths have been reported. Case fatality ratio in the US is around 1.8% (number of deaths/number of confirmed cases), in developing countries such as Brazil, the fatality ratio is around 2.7%, while in Mexico it goes as high as 9.2%.
Simple, inexpensive, and accurate diagnostic techniques are of utmost importance to isolate infected individuals and slow down the transmission of the disease, prevent oversaturation of health care facilities and attenuate morbidity and mortality. Accordingly, the scientific community has made remarkably quick strides to develop diagnostic tools, either for use in specialized health centers or for point-of-care community outposts.
Testing for SARS-CoV-2, the causal agent of COVID-19, is usually based on detecting proteins (viral antigens or host antibodies) or viral nucleic acids. Antibody detection tests indicate if the person has been infected by SARS-CoV-2 and has generated IgG and/or IgM antibodies. These tests are performed in blood serum or plasma and, while inexpensive and easy to administer, do not indicate if the infection is active , as it can take from 1 to 3 weeks after exposure to produce enough antibodies to be detected.
Antibody titers vary in patients; those that present milder symptoms or who are asymptomatic usually have relatively low antibody titers that disappear a few weeks after infection, while patients with more severe symptoms generally present higher antibody titers that may be detected two or three months after infection. When performed in the correct stage of infection antibody test sensitivity may be around 90% and results may be obtained in as little as 15 minutes.
Viral load-based tests detect viruses present in the host and can be either antigen-based, detecting specific fragments of viral proteins, or PCR-based, amplifying viral RNA. Unlike serological tests, these tests indicate if the patient has an active infection regardless of their immune response. Immunochromatographic antigen tests can yield results in 15 minutes. However, reported results range from 100% (based on 7 samples) to 32% accuracy (based on 106 positive RT-qPCR samples) .
The current FDA-recommended method to determine COVID-19 infections is based on reverse transcription quantitative polymerase chain reaction (RT-qPCR). This approach to virus detection amplifies specific sequences from viral SARS-CoV-2 RNA found in a given sample. Depending on the manufacturer, the nature and volume of the sample, and the oligonucleotides, RT-qPCR tests can detect as few as 242 SARS-CoV-2 RNA copies/mL or from 1 to 10 genomic copy equivalents per reaction .
There are three issues regarding standard RT-qPCR that make it less than ideal for large scale testing. First, the tests are usually performed using nasopharyngeal (NP) samples suspended in virus transport medium (VTM). As the sampling method is unpleasant, requires specialized swabs, and is difficult to self-administer, saliva sampling has been considered as an alternative source of specimens . Second, extraction of RNA from the samples is tedious and adds considerable time and expense to the assay. And third, RT-qPCR tests generally require expensive kits and access to an expensive thermocycler that may not be available in all settings. We sought to address all three of these issues to develop a faster, less expensive, and more accessible testing platform for detection of SARS-CoV-2 RNA from patients.
Residual samples were retained in a de-identified fashion with no link to patient identifiers. These remnant diagnostic swab samples from Fox Chase Cancer Center, Jeanes Hospital, and Temple University Hospital patients were stored in VTM at -80 °C after testing in the Fox Chase Molecular Diagnostics Laboratory. Saliva samples were obtained from healthy, consenting adult volunteers and stored at -80°C after pH measurments and RT-LAMP testing.
The SARS-CoV-2 diagnostic test used in the Fox Chase Molecular Diagnostics Laboratory extracts RNA from patient nasopharyngeal samples in VTM using a Qiagen QIAamp Viral or a Perkin Elmer chemagen Viral 300 kit, followed by RT-qPCR in an ABI QuantStudio 12K Flex instrument using the ThermoFisher TaqPath COVID-19 Combo Kit, that can detect at least 10 copies of virus per reaction. SARS-CoV-2 is stable and can be detectable by RT-qPCR and LAMP in both VTM and saliva for 7 to 25 days at a range of 4 to 30°C.
Protocols for nasopharyngeal (NP) and saliva samples
100X of inactivation buffer (0.5 M of TCEP-HCl, 0.1 M EDTA< pH 8, plus 1.15 N of NaOH, 0.1% μL of NP-10 and 5% of SDS in MQ water, pH 8 with NaOH) was added to treat the samples using the direct assay. Limit detection curves were made using diferent dilutions from the TaqPath COVID-19 RNA control, A47814 ThermoFisher Scientific. Samples were immediately vortexed, pulse-centrifuged, incubated at 95°C for 5 minutes and centrifuged 30s at 5,000 xg to precipitate the proteins in the VTM and saliva samples. The addition of detergents and heating ensures killing the virus. 1.0 μL of this supernatant was added to a previously set up 10 μL RT-LAMP reaction.
RNA precipitation assay.
Nucleotides present in the sample were precipitated using silica beads . Briefly, NP samples in VTM or saliva were added to an Eppendorf tube containing a solution with 100X inactivation buffer, and RNAsecure (25X) . The addition of the RNAsecure (Beta-mercaptoethanol mix), irreversibly denatures RNAses by reducing disulfide bonds therefore protecting RNA. For saliva samples, one microliter of proteinase K (MEB 8107S) 1:10 dilution was added per 250 μL reaction. Samples were vortexed, pulse-centrifuged, incubated at 55°for 15 min and 95°C for 5 minutes, and centrifuged 30s at 5,000 xg to precipitate the unwanted protein. Treated samples were transferred to a new tube, taking care to avoid carry over of the precipitate.
We added 0.35 mL of RNA binding solution (6M of NaI, 2% Triton-100 and 10 mM HCl) and 5 μL of glass milk/silica gel 1:1 w/v in 10 mM Tris-HCl pH 8 and 1 mM EDTA pH 8, per 0.75 mL of sample and left at room temperature during 15–20 minutes shaking carefully by inversion every two minutes. Samples were centrifuged 1 min at maximum speed in a microcentrifuge. The supernatant was discarded in 10% bleach and the pellet was washed with 80% EtOH without dislodging it. Samples were centrifuged for 1 min at 13,000 xg, then ethanol was discarded and tubes were dried at 55°C for one minute. Samples were resuspended in 9 μL of preheated 1x inactivation buffer and used for the RT-LAMP assay or kept at -80°C. 3 μL of this sample were added directly to a previously set up 10 μL RT-LAMP reaction.
Reactions were set up according to the WarmStart LAMP Kit (NEB). First the LAMP master mix was added to the PCR tubes to avoid contamination. We used two or three sets of oligos for each assay: NEB Gene N-A, HMS Assay 1e, NEB orf1a-A oligonucleotides, and an actin control (ACTB) for saliva samples. Primers were designed for specific genes from the genome of the SARS-CoV-2.