One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

Abstract

Introduction: Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR.
 Methodology: The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3´ end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR.
 Results: Amplification was achieved for the positive control and specific regions in both techniques.
 Conclusions: This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.

Patient-Reported Health Outcomes After Treatment of COVID-19 with Nebulized and/or Intravenous Neutral Electrolyzed Saline Combined with Usual Medical Care Versus Usual Medical care alone: A Randomized, Open-Label, Controlled Trial.

“…In 10 experimental group patients treated with electrolyzed saline, nasopharyngeal and oropharyngeal samples were collected with swabs in 2.5 ml of viral transport medium, right before starting treatment and on days 2, 4, 6, and 14, and stored at -80 °C until processing. Viral RNA was isolated utilizing TRIzol (Invitrogen, Carlsbad CA, USA), following the manufacturer's instructions, and SARS-CoV-2 testing was carried out through the SYBR Green-Based Real-Time RT-PCR, using the previously described methodology [43]. That procedure was not carried out on any of the control group patients.…”

Safety and efficacy of a COVID‑19 treatment with nebulized and/or intravenous neutral electrolyzed saline combined with usual medical care vs. usual medical care alone: A randomized, open‑label, controlled trial

“…In 10 patients from the experimental group treated with electrolyzed saline, nasopharyngeal and oropharyngeal samples were collected with swabs in 2.5 ml of viral transport medium, immediately prior to starting treatment and on days 2, 4, 6 and 14, and stored at -80˚C until processing. Viral RNA was isolated utilizing TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and SARS-CoV-2 testing was performed through SYBR green-based reverse transcription-quantitative (RT-q) PCR using the previously described methodology ( 59 ). That procedure was not performed on any of the patients in the control group.…”

An undergraduate laboratory experiment with real‐world applications: Utilizing templateless polymerase chain reaction and real‐time polymerase chain reaction to test for SARS‐CoV‐2 RNA

“…A synthetic, non-infectious sequence was generated to serve as a positive control, based on a paper by Meza-Robles et al, 22 using templateless PCR to create a large positive control sequence based on the SARS-CoV2 ORF1ab gene sequence. Three oligonucleotides (1COV, 2COV, 3COV) were synthesized (Table S1) and assembled by templateless PCR.…”

“…The synthetic positive control was based on a procedure by Meza-Robles et al for onestep nested SARS-CoV-2 RT-PCR. 22 This exercise utilizes no virus, so it involves no additional safety concerns. Pedagogical advantages of the experiment include problembased learning, experimental design, clinical applications and a real-world application.…”