Due to the lack of protective immunity in the general population and the absence of effective vaccines and antivirals, the Coronavirus disease 2019 (COVID-19) pandemic continues in some countries, with local epicentres emerging in others. Due to the high demand for effective COVID-19 testing programs to control the spread of the disease, we have suggested such a testing program that includes a rapid RT-qPCR approach without RNA extraction. The Direct-One-Step-RT-qPCR (DIOS-RT-qPCR) assay detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one hour while maintaining the high sensitivity and specificity required of the testing tools diagnosis.
This optimized protocol allows the direct use of swab transfer media (14 μL) without the need for RNA extraction, achieving sensitivity comparable to the standard method that requires the costly and time-consuming step of RNA isolation. The detection limit for DIOS-RT-qPCR was less than seven copies/reaction, which translates to 550 virus copies/ml of the swab. The speed, ease of use, and low price of this assay make it suitable for high-throughput screening programs. The use of fast enzymes allows RT-qPCR to be performed under standard laboratory conditions in one hour, making it a possible point-of-care solution in high-speed cycling instruments.
This protocol also implements the heat inactivation of SARS-CoV-2 (75 ° C for 10 min), rendering the samples non-infectious, allowing testing at the BSL-2 facility. Additionally, we discuss the critical steps involved in developing tests for the rapid detection of COVID-19. Implementing quick, easy, and cost-effective methods can help control the global spread of the COVID-19 infection.
2019-nCoV; diagnostics; One-step direct RT-PCR; screening tests; testing at the point of care.
Materials and Methods
1. LAMP reactions
Fluorescent LAMP reactions were performed using the WarmStart LAMP kit (E1700L, New England Biolabs, NE, USA), and the WarmStart LAMP 2X colourimetric master mix (M1800L, New England Biolabs, NE, USA) was used. for colorimetric LAMP. All LAMP reactions were carried out following the manufacturer’s instructions, for every 29 μL of reaction we used 2.5 μL of a mixture of LAMP primers (F3, B3: 2 μM, LF, LB: 4 μM and FIP, BIP: 16 μM), 5 µL of sample or control and the concentration of MgSO4 was increased to 6 mM by adding 1.13 µL per reaction, of a 100 mM solution.
For the colourimetric and fluorescent LAMP reactions, amplification was carried out by incubating at 65 ° C for 40 min. For real-time fluorescent detection of LAMP reactions, we used a thermal cycler of the QuantStudio 3 real-time PCR system (ThermoFisher Scientific, MA, USA), using a PCR program of 160 cycles of 15 s each. End-point fluorescence quantification was carried out on a Beckman Coulter DTX 880 multimode detector (Beckman Coulter, Nyon, Switzerland), using the FAM excitation and emission filters. Direct visualization of fluorescence was performed on a benchtop transilluminator.
2. LbCas12a detection
For LbCas12a-mediated detection, we used commercial LbaCas12a (Engen LbaCas12a, M0653T, NEB, NE) and RNA was synthesized as RNA oligonucleotides by IDT (CA, USA). First, the LbaCas12a-gRNA complexes were generated by incubating LbaCas12a (50 nM, final concentration) and gRNA (62.5 nM), in 1X NEBuffer 2.1 at a final volume of 20 µL per reaction. The LbaCas12a-gRNA complexes were incubated for 30 min at 37 ° C, after incubation, the Taqman probe was added to a final concentration of 500 nM and transferred to ice.
After completion of the LAMP reaction, 2 µL of each LAMP reaction was added to 20 µL of the LbaCas12a-gRNA complexes, and the reactions were incubated at 37 ° C for 10 min. Real-time fluorescence acquisition was performed by the thermal cycler of the QuantStudio 3 Real-Time PCR system (ThermoFisher Scientific, MA, USA), using a qPCR program of 80 cycles of 15 s at 65 ° C. Visual inspection Fluorescence testing was performed using a blue light transilluminator (Invitrogen, CA, USA).
3. In silico alignment
We used blastn from NCBI for in silico alignment, we compared each primer sequence to the main virus and bacteria that cause symptoms similar to covid-19 or are related to the SARS-CoV-2 virus (Candida albicans taxid: 5476, Neisseria meningitides taxid: 487, Pseudomonas aeruginosa taxid: 287, Staphylococcus aureus taxid: 1280, Influenza A taxid: 11320, Influenza B taxid: 11520, MERS bat coronavirus taxid: 2664423, MERS-CoV taxid: 1335626, HCoV-SARS tax: 694009, Tax ID HCV-229E: 1113
4. ROC curves and AUC values
The receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) values were calculated using the pROC R package (1.16.2) with the cycle threshold (Ct) values from the LOD curves. o the evaluation of patient samples from fluorescent LAMP reactions or the qualitative values of the use of the Cas12a variation. Non-amplified cases were considered as Ct = 160. A 50% AUC is related to a positive result by chance, while we consider a percentage greater than 90% as acceptable and an exact result.
5. Sample processing and RNA extraction
For LOD determination, we added a known number of RNA copies of the N gene to 8 μL of a healthy donor nasal swab, and subsequently added 2 uL of 5X lysis solution (0.5 M DTT and 5 mM EDTA). The samples were incubated at 42 ° C for 20 min, followed by 64 ° C for 5 min. These steps allowed nuclease inactivation and lysis of biological material releasing nucleic acid without extraction. Five microliters of these inactivated samples were added to 20 µL of LAMP reaction and processed as specified above. Furthermore, this simple inactivation method was used for virus inactivation and lysis of patient samples.
The Institutional Review Board (IRB) of the National Institute of Respiratory Diseases (INER) reviewed and approved the protocols for COVID-19 studies. This project was approved by the IRB under registration number B09-20. All subjects or their legal guardians, particularly in the case of critically ill patients, gave their written informed consent for these studies and authorized the storage of their nasopharyngeal samples in the INER repositories for this and future studies. In this study, we did not collect samples from minors/children, only young adults older than 17 years were included.
A total of 34 patient samples were collected in the Molecular Biology Unit of Molecular Biology of the National Institute of Respiratory Diseases of Mexico City. We collected 31 nasal swabs and 3 saliva samples from individuals who attended the institute emergency room between March 1 and May 31, 2020; coinciding with the increase in the COVID-19 outbreak in Mexico City. RNA extraction was performed by adding 2 μL of lysis solution (0.5 M DTT and 5 mM EDTA) to 8 μL of the sample, followed by incubation at 42 ° C for 20 min and 64 ° C for 5 min.
For the LAMP reactions, we used 5 μL of the extracted samples for the amplification of the SARS-CoV-2 N gene and 5 μL for the amplification of POP7. Real-time fluorescence measurement was performed using a StepOnePlus real-time PCR system (ThermoFisher, MA, USA) for RT-qPCR and LAMP reactions. The presence of viral transcription was first assessed by one-step RT-PCR with an internally standardized modified Berlin method, using specific oligonucleotide sequences for the E and RdRp genes (2019-nCoV) and confirmed with the LAMP test.