Single enzyme RT-PCR of full-length ribosomal RNA

Hammerling, Michael J.; Yoesep, Danielle J.; Jewett, Michael C.

doi:10.1093/synbio/ysaa028

Cited by 3 publications

(2 citation statements)

References 56 publications

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“…Although RT-qPCR-based SARS-CoV-2 diagnostic tools are generally considered the gold standard for disease detection, several studies have determined that this approach is prone to return false-negative results due to gene mutations (which is often the case for the E and N genes) Tahan et al, 2021) or primer dimer formation (Jaeger et al, 2021). Another factor that affects RT-qPCR efficiency is the secondary structure of the RNA to be characterized/quantified (Hammerling et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Cadena-Caballero,

Vera-Cala,

Barrios-Hernandez

et al. 2024

F1000Res

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Background The COVID-19 pandemic, caused by the SARS-CoV-2, can be effectively managed with diagnostic tools such as RT-qPCR. However, it can produce false-negative results due to viral mutations and RNA secondary structures from the target gene sequence. Methods With High Performance Computing, the complete SARS-CoV-2 genome was obtained from the GenBank/GISAID to generate consensus sequences to design primers/probes for RT-qPCR. ORF8 gene was selected, meanwhile, E and N and RNAse P were according to CDC protocol. Nasopharyngeal swab samples were collected from patients diagnosed with SARS-CoV-2. Total RNA was purified according MagMAX kit, it was used in single, and multiplex RT-qPCR. To avoid templated secondary structures, compensate nucleotide proportions and improve Ct values, a solution composed of tetraethylammonium chloride and dimethyl sulfoxide and other with corresponding to dNTPs proportions in accordance SARS-CoV-2 genome were included. Sensitivity and specificity according to Ct values were determined with the Caret package in R software. Results 126,576 SARS-CoV-2 genomes from January to December 2020 comprised a database. From this, a region near of 5′ ORF8 gene showed three stem-loops was used for primers/FAM-probe. 49 samples were obtained, from them, 22 were positive to gene selected regions. Interestingly, samples from October to November 2020 were positive for all regions, however, in January 2021 different results were observed in ORF8. An improvement in Ct with the adjuvant solutions was determined in all samples with others SARS-CoV-2 primers/probes, for both single and multiplex RT-qPCR. The inclusion of the denaturing solution in single RT-qPCR increased its sensitivity with respect to the commercial method, while in multiplex the opposite was generated. Conclusions Including adjuvant solutions to prevent the formation of RNA secondary structures and the adjustment of the nucleotide ratios of SARS-CoV-2 improved single and multiplex RT-qPCR for viral identification, demonstrating its potential application in health public.

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Section: Introductionmentioning

confidence: 99%

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Cadena-Caballero,

Vera-Cala,

Barrios-Hernandez

et al. 2024

F1000Res

View full text Add to dashboard Cite

show abstract

“…Although RT-qPCR-based SARS-CoV-2 diagnostic tools are generally considered the gold standard for disease detection, several studies have determined that this approach is prone to return false-negative results due to gene mutations (which is often the case for the E and N genes) (Hasan et al, 2021;Tahan et al, 2021) or primer dimer formation (Jaeger et al, 2021). Another factor that affects RT-qPCR efficiency is the secondary structure of the RNA to be characterized/quantified (Hammerling et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

et al. 2022

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Background: Recent estimates indicate that the COVID-19 pandemic, which is caused by the SARS-CoV-2 virus, could be effectively controlled via the development and implementation of diagnostic tools such as quantitative reverse transcription PCR (RT-qPCR). However, this reaction often generates false-negative results due to novel mutations and can also be affected by the secondary structure of the RNA transcripts that derive from the gene sequence used for diagnostic purposes. Methods: Using high-performance computing, we consolidated a global SARS-CoV-2 genome repository encompassing 19,317 genomes from the GenBank database and 107,259 from the GISAID database to generate monthly SARS-CoV-2 consensus sequences from January to December 2020. Results: These sequences were then used to create ORF8-specific primers and probes to validate single and multiplex RT-qPCR protocols both in silico and experimentally using genes E (Berlin protocol) and N (CDC protocol) as targets. Conclusions: Our findings demonstrated that RT-qPCR Ct values were improved by the inclusion of either a denaturing solution composed of tetraethylammonium chloride (TEA) and dimethyl sulfoxide (DMSO) and by adjusting nucleotide proportions based on the SARS-CoV-2 genome.

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Salt, Fat, Acid, Heat: Modulators of Signal Transduction

Favichia

Gibney

Kowalewski

et al. 2022

Encyclopedia of Life Sciences

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Signal transduction pathways are heavily dependent on interactions with the cellular environment. The same environmental elements that make food tasty (salt, fat, acid, heat) have profound impacts on many other aspects of protein function. Transducing sensory information involves a change in receptor confirmation on a sensory neuron/end organ (e.g. taste bud), which allows perception of salt, fat, acid and heat. Beyond our perception, however, are the wide‐ranging effects that these environmental elements have on the intracellular signal transduction pathways which impacts every aspect of cell, tissue, organ and organism function. The proteins constituting signalling pathways are constantly receiving environmental information including ionic charges (salt), hydrophobicity (fat), protonation state (acid) and thermal energy (heat). Key Concepts Signal transduction pathways can be altered by environmental fluctuations. Salts dissociate into their separate ionic forms. Regulating ion type and concentration are important for maintaining protein structure, enzyme activity, protein interactions and controlling ion channels. Lipid modifications can influence a protein's microdomain localisation to its target cell and impact ease of intracellular or extracellular diffusion. Fluctuations in pH alter the protonation state of proteins and ion channels which can impact its specificity, affinity to binding partners, structural dynamics and the pathways that are activated. Temperature changes (e.g. extreme hot or cold) can inactivate and denature proteins generally but specifically may gate temperature‐sensitive ion channels [i.e. transient receptor potential ( TRP ) channels]. Changes in temperature can also reveal gain or loss of function phenotypes for mutations that confer less severe phenotypes at physiological temperatures, as exemplified by numerous temperature‐sensitive alleles used in genetics and cell biology experiments.

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Single enzyme RT-PCR of full-length ribosomal RNA

Cited by 3 publications

References 56 publications

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Salt, Fat, Acid, Heat: Modulators of Signal Transduction

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