A Rapid SARS-CoV-2 RT-PCR Assay for Low Resource Settings
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Abstract
Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.
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SciScore for 10.1101/2020.04.29.069591: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Alternatively, software such as ImageJ can be utilized. ImageJsuggested: (ImageJ, RRID:SCR_003070)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:The only limitation we expect for this set up is when using it in high altitude locations where boiling temperature …
SciScore for 10.1101/2020.04.29.069591: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Alternatively, software such as ImageJ can be utilized. ImageJsuggested: (ImageJ, RRID:SCR_003070)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:The only limitation we expect for this set up is when using it in high altitude locations where boiling temperature is lower than 95°C. Lower denaturation temperature setting would be needed, along with longer denaturation time. RT-qPCR analysis of ten COVID-19 positive clinical samples: To determine the relative viral load of the clinical samples, 60 μL of the specimen was extracted using an automated system (Promega Maxwell) and run on a commercial real-time thermal cycler. Three μL of the templates were used in each RT-qPCR reaction. The qPCR threshold cycle (Ct) values of these samples are listed in Table 1. It took the commercial cycler 1 hour and 22 minutes to complete the reaction. Furthermore, to test the need for RNA extraction, 3 μLs of each media was spiked in 17 μL of the master mix. The results show that untreated media samples can produce qualitative PCR results matching those performed using extracted RNA templates. Rapid RT-PCR detection of SARS-CoV-2 with N1, N2, and RNase P reactions using water baths: To test for SAR-CoV-2 using water baths, we prepared three singleplex PCR reactions, each targeting N1, N2, and RNase P. Extracted templates from COVID-19 positive clinical specimens and contrived negative samples were tested using water bath-based RT-PCR. The RT-PCR run was completed in 12 minutes. The picture of the PCR tubes after 40 cycles shows that we can use a blue LED gel box and cell phone camera to determine the test results (Figure 2). In a COVID-19...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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