Development of an optimized colorimetric RT-LAMP for SARS-CoV-2 assay with enhanced procedure controls for remote diagnostics

Abstract

The coronavirus pandemic accentuated the need for molecular diagnostic tests. A technique highly used to this end is the Polymerase Chain Reaction (PCR)—a sensitive and specific technique commonly used as the gold standard for molecular diagnostics. However, it demands highly trained personnel and high-maintenance equipment and is relatively time-consuming. An alternative is the Loop-Mediated Isothermal Amplification (LAMP) technique, which doesn’t need sample purification or expensive equipment, and is similar to PCR when compared in sensitivity and specificity. In this paper, we developed an optimized colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) Point-of-Care test using a portable device to diagnose COVID-19. Variables such as concentration of primers, magnesium sulfate, betaine, hydrochloride guanidine, Bst, and temperature of the reactions were tested. We also created a pipetting quality control system—using a combination of dyes—to avoid false negatives due to a lack of samples added to the reaction test tube. Mineral oil was incorporated in the composition of the RT-LAMP reactions to avoid evaporation when a heating lid isn't available. The final RT-LAMP test is tenfold more sensitive when compared to the WarmStart Colorimetric Master mix from New England Biolabs with a sensitivity of 5 copies per μL.

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system

“…For the E. coli DNA contamination assay, three sets of primers targeting the E. coli ribosome 16S gene were used. Two of them (sets 1 and 2) were obtained from the literature, 39,40 and the other (set 3) was designed using the Primer-BLAST tool from NCBI. All three sets amplified the target gene accordingly in the PCR reactions (Supplemental Figure S5(A)).…”

“…For CQMED Bst -LF optimal concentration determination, LAMP reactions were set up with 10,000 copies per reaction of pUC17 plasmids with the SARS-CoV-2 regions of interest (N2 and E1) diluted in the sample solution. NEB Bst 2.0 WarmStart enzyme was used as a reference in the previously optimized concentration of 0.4 U/μL, 40 and CQMED Bst -LF concentration varied from 4 to 12 ng/μL, both diluted in Diluent A to 100×, so the concentration of the enzyme buffer remains the same in all reactions.…”

Development of loop-mediated isothermal amplification (LAMP) assays using five primers reduces the false-positive rate in COVID-19 diagnosis

“…Accordingly, based on primers’ melting temperature (Tm) and after optimizing reaction conditions, 40–60 min at 70 °C were the optimum settings. Raddaz et al 37 also optimized their RT-LAMP assay by testing varying concentrations of the reaction components, such as Bst DNA polymerase, primers, and MgSO 4 concentrations, which significantly enhanced the sensitivity up to ten folds compared to the WarmStart protocol without optimization. Unlike our study, GuHCl addition had no effect in improving time to reaction (TTR) response in their assay since it was a duplex (targeting both N and ORF1ab genes), which supports the idea that the effect of GuHCl varies depending on the primers used.…”

Evolution and Impact of Nucleic Acid Amplification Test (NAAT) for Diagnosis of Coronavirus Disease

“…112 Hologic’s Aptima SARS-CoV-2 Assay employs TMA, while direct RNA amplification requires reverse transcriptase with the T7 RNA polymerase enzyme. Several authorized NATs based on TMA use the Aptima SARS-CoV-2 Assay, which is the Poplar SARS-CoV-2 TMA Pooling Assay, 113 the Quest Diagnostics HA SARS-CoV-2 Assay, 114 the Let us Get Checked Coronavirus Assay (COVID-19) TEST, 115 and the Procleix SARS-CoV-2 Assay, 116 which detects ORF1ab genes in two regions and an IC, which is intended to be carried out using the Panther System. The target RNA molecule is extracted with the help of a capture oligomer with a sequence complementary to the target protein’s specific region and a chain of deoxyadenosine residues.…”