A One-Step open RT-qPCR for SARS-CoV-2 detection

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), etiological agent of the coronavirus disease 2019 (COVID-19), is currently detected by reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) of its viral RNA genome. Within the available alternatives, One-Step procedures are preferred since they are fast and significantly decrease preanalytical errors, minimizing the risk of diagnostic errors.Increasing the testing capacity and tracing contacts are essential steps to control the pandemic. However, high-cost commercial reagents subject to shortage and poor scalability have hindered the use of these technologies and their adoption for a wide population-scale testing, being even more critical in developing countries. In the current context, open-source initiatives have promoted global collaboration to promote accessible solutions for rapid local deployment. As a result, open protocols are being developed for the local production of SARS-CoV-2 diagnostics.This work aimed to produce an open-source system for SARS-CoV-2 diagnostic tests in RNA clinical samples. We provide guidelines for standardizing an open One-Step RT-qPCR master mix using recombinant M-MLV reverse transcriptase together with either Pfu-Sso7d or Taq DNA polymerase. Both were tested on synthetic RNA and clinical samples, observing a good correlation when compared to commercial RT-qPCR kits. Nevertheless, the best results were obtained using M-MLV RT combined with Taq DNA polymerase in a probe-based RT-qPCR assay, allowing successful discrimination between positive and negative samples with accuracies comparable to a CDC-recommended commercial kit.Here, we demonstrate that these open RT-qPCR systems can be successfully used to identify SARS-CoV-2 in clinical samples and potentially be implemented in any molecular diagnostic laboratory.

Modification Of Taq DNA Polymerase with Improved Elongation Ability

Thermostable polymerases play a significant role in molecular biology and diagnostic practice. The most famous and demanded is Polymerase I from the thermophilic bacterium Thermus aquaticus (Taq-pol). This polymerase at one time made a kind of revolution in the polymerase chain reaction. In this work, we attempted to modify this polymerase by attaching an additional Sso7d protein from Sulfolobus solfataricus to Taq-pol, which provides additional binding to the double-stranded DNA of the template. Sso7d-Taq fusion gene was expressed in BL21(DE3) cells. Optimal conditions were selected for maximum production of modified Sso7d-Taq polymerase. The optimal conditions for the intracellular accumulation of Sso7d-Taq polymerase: activation of the T7 promoter when the optical density of the culture reaches OD600 = 0.8-1.0 by adding IPTG at a concentration of 0.2 mM, followed by incubation of the culture at 37°C for 20-24 hours. Recombinant Sso7d-Taq polymerase has been purified and tested by PCR for thermal stability and elongation time. It was found that the Sso7d-Taq enzyme withstands 5 hour incubation at 95°C and 75 minute incubation at 98°C. Comparative analysis with unmodified Taq DNA polymerase showed that the Sso7d-Taq enzyme reduces the elongation rate by several times - up to 15-13 seconds per 1 kbp. The results obtained indicate the prospects of using Sso7d-Taq DNA polymerase in scientific research and diagnostic practice.

Recombinant protein expression and purification of Taq DNA polymerase v1

This is a slightly modified and simplified version of a protocol by Thomas G.W. Graham et al, which is available at https://gitlab.com/tjian-darzacq-lab/bearmix and has been described in depth in the article 10.1371/journal.pone.0246647, for the recombinant expression of a E602D mutant of Taq DNA polymerase in pET-28a that is available in Addgene (Addgene plasmid # 166944 ; http://n2t.net/addgene:166944 ; RRID:Addgene_166944). The main goal of this protocol is to eliminate the use of large volumes for dialysis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions.

Development of a recombinant Taq DNA polymerase enzyme expressed using a synthetic gene and its comparison with a commercial enzyme

Taq DNA polymerase is a thermostable enzyme widely used for DNA amplification in the PCR technique. It was initially characterized and isolated from thermophilic bacteria, Thermus aquaticus. It was difficult to developed in this enzyme using a native host system. Therefore, the development of the recombinant Taq DNA polymerase expressed using a synthetic gene is important to improve production efficiency. In this study, we developed the in house Taq DNA polymerase recombinant based on a codon-optimized using E. coli expression system. We cloned 2685 bp of the Taq DNA polymerase gene in the pET151/D-TOPO vector. The gene was synthesized and the expression was analyzed with SDS-PAGE technique which indicated with a 100.9 kDa specific target protein. The concentration and activity of this purified enzyme were 5.17 mg/mL and 4.647 U/µL, respectively. The application of this enzyme to the PCR technique showed that this enzyme could amplify the target genes from 200 bp to 3500 bp amplicons with a minimum DNA concentration template 10 ng/µL. This assumes that the in house recombinant Taq DNA polymerase based on synthetic genes is successfully expressed, purified, and was functional and comparable to the commercial Taq polymerase.

Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens

A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.

A Single Amino Acid Change to Taq DNA Polymerase Enables Faster PCR, Reverse Transcription and Strand-Displacement

A change of an aspartic acid to asparagine of Taq (Thermus aquaticus) DNA polymerase is a gain of function mutation that supports faster PCR: the extension times for PCR amplification can be 2–3 times shorter. Surprising results from negative controls led to the discovery of strand-displacement ability and reverse transcriptase activity of Taq D732N DNA polymerase. We demonstrate that the mutant enzyme can, by itself, catalyze RT-PCR, and RT-LAMP assays. Residue 732 is on the surface of the enzyme, not near the active site.