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Scientific Reports Jan 2024mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP)...
mRNA measurement is dominated by RT-PCR, which requires expensive laboratory equipment and personnel with advanced degrees. Loop-mediated isothermal amplification (LAMP) is a versatile technique for detecting target DNA and RNA. The sensitivity of LAMP in early reports has been below that of the standard RT-PCR tests. Here, we report the use of a fluorescence-based RT-LAMP protocol to measure CDX2 expression patterns, which match extremely well to the standards of sophisticated RT-PCR techniques (r = 0.99, p < 0.001). The assay works on diverse sample types such as cDNA, mRNA, and direct tissue sample testing in 25 min compared to more than 3 h for RT-PCR. We have developed a new protocol for designing RT-LAMP primers that reduce false positives due to self-amplification and improve quantification. A simple device with a 3D-printed box enables the measurement of mRNA expression at home, outdoors, and point-of-care setting.
Topics: RNA, Messenger; RNA; Biological Assay; DNA Primers; DNA, Complementary
PubMed: 38200031
DOI: 10.1038/s41598-023-49651-8 -
Transboundary and Emerging Diseases Mar 2021To combat the COVID-19 pandemic, millions of PCR tests are performed worldwide. Any deviation of the diagnostic sensitivity and specificity will reduce the predictive...
To combat the COVID-19 pandemic, millions of PCR tests are performed worldwide. Any deviation of the diagnostic sensitivity and specificity will reduce the predictive values of the test. Here, we report the occurrence of contaminations of commercial primers/probe sets with the SARS-CoV-2 target sequence of the RT-qPCR as an example for pitfalls during PCR diagnostics affecting diagnostic specificity. In several purchased in-house primers/probe sets, quantification cycle values as low as 17 were measured for negative control samples. However, there were also primers/probe sets that displayed very low-level contaminations, which were detected only during thorough internal validation. Hence, it appears imperative to pre-test each batch of reagents extensively before use in routine diagnosis, to avoid false-positive results and low positive predictive value in low-prevalence situations. As such, contaminations may have happened more widely, and COVID-19 diagnostic results should be re-assessed retrospectively to validate the epidemiological basis for control measures.
Topics: Benchmarking; COVID-19; COVID-19 Testing; DNA Primers; Equipment Contamination; Germany; Humans; Pandemics; Real-Time Polymerase Chain Reaction; SARS-CoV-2; Sensitivity and Specificity
PubMed: 32536002
DOI: 10.1111/tbed.13684 -
Scientific Reports Jun 2022DNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε binds two substrates, a template:primer and dNTP, and...
DNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε binds two substrates, a template:primer and dNTP, and catalyzes a covalent attachment of dNMP to the 3' end of the primer. Previous studies have shown that Polε easily inserts and extends ribonucleotides, which may promote mutagenesis and genome instability. In this work, we analyzed the mechanisms of discrimination against RNA-containing primers by human Polε (hPolε), performing binding and kinetic studies at near-physiological salt concentration. Pre-steady-state kinetic studies revealed that hPolε extends RNA primers with approximately 3300-fold lower efficiency in comparison to DNA, and addition of one dNMP to the 3' end of an RNA primer increases activity 36-fold. Likewise, addition of one rNMP to the 3' end of a DNA primer reduces activity 38-fold. The binding studies conducted in the presence of 0.15 M NaCl revealed that human hPolε has low affinity to DNA (K of 1.5 µM). Strikingly, a change of salt concentration from 0.1 M to 0.15 M reduces the stability of the hPolε/DNA complex by 25-fold. Upon template:primer binding, the incoming dNTP and magnesium ions make hPolε discriminative against RNA and chimeric RNA-DNA primers. In summary, our studies revealed that hPolε discrimination against RNA-containing primers is based on the following factors: incoming dNTP, magnesium ions, a steric gate for the primer 2'OH, and the rigid template:primer binding pocket near the catalytic site. In addition, we showed the importance of conducting functional studies at near-physiological salt concentration.
Topics: DNA; DNA Polymerase II; DNA Primers; DNA Replication; Humans; Kinetics; Magnesium; Nucleotides; Templates, Genetic
PubMed: 35715491
DOI: 10.1038/s41598-022-14602-2 -
Chembiochem : a European Journal of... Sep 2020DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional...
DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional secret keys in DNA steganography. Brute force testing of different primers will be extremely time consuming, and practically unaffordable when high-throughput sequencing is used. However, the encrypted information can be sequenced and read once the primers are intercepted. A new steganography approach is needed to make the DNA-encoded information safer, if not unhackable. Mixing information-carrying DNA with a partially degenerated DNA library containing single or multiple restriction sites, we have built an additional protective layer that can be removed by desired restriction enzymes as secondary secret keys. As PCR is inevitable for reading DNA-encrypted information, heating will cause reshuffling and generate endonuclease-resistant mismatched duplexes, especially for DNA with high sequence diversity. Consequently, with the incorporation of randomness, DNA steganography possesses both quantum key distribution (QKD)-like function for detecting PCR by an interceptor and a self-destructive property. It is noteworthy that the background noise generated through the protective layer is independent from any sequencing technology including Sanger and high-throughput sequencing. With a DNA ink incorporating the steganography, we have shown that the authenticity of a piece of writing can be confirmed only by authorized persons with knowledge of all embedded keys.
Topics: Computer Security; DNA; DNA Primers; Humans; Polymerase Chain Reaction
PubMed: 32270906
DOI: 10.1002/cbic.202000149 -
Scientific Reports Apr 2021Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into...
Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.
Topics: DNA Primers; DNA Probes; Genome, Viral; Mutation; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2
PubMed: 33903676
DOI: 10.1038/s41598-021-88532-w -
Clinical Biochemistry Oct 2020A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019...
OBJECTIVES
A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks.
METHODS
Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration.
RESULTS
We found that the freeze-dried PCR mixes with ~1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18 ~ 25 °C) for 28 days, and for 14 and 10 days when stored at 37 °C and 56 °C, respectively.
CONCLUSION
The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories.
Topics: Betacoronavirus; COVID-19; COVID-19 Testing; Clinical Laboratory Techniques; Coronavirus Infections; DNA Primers; DNA, Viral; Freeze Drying; Humans; Pandemics; Pneumonia, Viral; Polymerase Chain Reaction; SARS-CoV-2; Temperature
PubMed: 32592724
DOI: 10.1016/j.clinbiochem.2020.06.013 -
Nucleic Acids Research Feb 2023DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in...
DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme. Using novel HelQ mutant proteins we identify that inhibition of Pol δ is independent of DNA binding, and maps to a 70 amino acid intrinsically disordered region of HelQ. Pol δ and its POLD3 subunit robustly stimulated DNA single-strand annealing by HelQ, and POLD3 and HelQ interact physically via the intrinsically disordered HelQ region. This data, and inability of HelQ to inhibit DNA synthesis by the POLD1 catalytic subunit of Pol δ, reveal a mechanism for limiting DNA synthesis and promoting DNA strand annealing during human DNA break repair, which centres on POLD3.
Topics: Humans; DNA; DNA Polymerase III; DNA Primers; DNA Replication; Proliferating Cell Nuclear Antigen; DNA Helicases
PubMed: 36718939
DOI: 10.1093/nar/gkad032 -
Archives of Razi Institute Aug 2023Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the...
Since pebrine disease, as the most important and dangerous disease in silkworms, spreads horizontally through the spores and vertically through the eggs, combating the disease and eliminating it completely from livestock production has been associated with numerous problems. This project aimed to identify the molecular cause of pebrine disease in silkworms using a sensitive, specific, and accurate method. To this purpose, a 136 bp fragment was selected based on the partial SSU rDNA sequence, and a pair of primers was designed. Afterward, using the conventional polymerase chain reaction (PCR) method, the target fragment was amplified and sequenced. After that, to determine the detection sensitivity, using the Real-Time PCR method, 5-fold serial dilutions of DNA were prepared, and the last dilution that produced a fluorescent signal was considered the minimum detection limit. All tests were performed in duplicates. Based on the results of the sensitivity test, the standard curve including Ct values and DNA concentration was used for analysis. Moreover, 80 unknown samples examined by light microscope were evaluated using conventional PCR and Real-Time PCR. Both PCR results showed no amplification for the negative control samples. The findings demonstrated that the lowest detection limit for was less than 6 pg of DNA, while, this amount was 8 ng for conventional PCR. Out of 80 samples examined, 55, 60, and 62 samples were positive for light microscope, conventional PCR, and Real-Time PCR methods, respectively. The findings suggested that the Real-Time PCR method had a higher ability to detect the causative agent of pebrine disease than the conventional PCR method, and both methods were superior to light microscopy. Therefore, due to the fewer steps and higher accuracy of Real-Time PCR, it can be introduced as a suitable method for diagnosing pebrine disease.
Topics: Animals; Bombyx; Microsporidiosis; Real-Time Polymerase Chain Reaction; DNA Primers; DNA
PubMed: 38226388
DOI: 10.32592/ARI.2023.78.4.1185 -
Critical review of methods for isothermal amplification of nucleic acids for environmental analysis.Journal of Microbiological Methods Dec 2020The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic... (Review)
Review
The past 30 years have seen the emergence and proliferation of isothermal amplification methods (IAMs) for rapid, sensitive detection and quantification of nucleic acids in a variety of sample types. These methods share dependence on primers and probes with quantitative PCR, but they differ in the specific enzymes and instruments employed, and are frequently conducted in a binary, rather than quantitative format. IAMs typically rely on simpler instruments than PCR analyses due to the maintenance of a single temperature throughout the amplification reaction, which could facilitate deployment of IAMs in a variety of environmental and field settings. This review summarizes the mechanisms of the most common IAM methods and their use in studies of pathogens, harmful algae and fecal indicators in environmental waters, feces, wastewater, reclaimed water, and tissues of aquatic animals. Performance metrics of sensitivity, specificity and limit of detection are highlighted, and the potential for use in monitoring and regulatory contexts is discussed.
Topics: Animals; Bacteria; DNA Primers; Environmental Monitoring; Harmful Algal Bloom; Nucleic Acid Amplification Techniques; Nucleic Acids; Real-Time Polymerase Chain Reaction; Schistosoma; Wastewater; Water Microbiology
PubMed: 33159993
DOI: 10.1016/j.mimet.2020.106099 -
Nature Aug 2022The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication...
The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.
Topics: DNA; DNA Primase; DNA Primers; DNA Replication; Humans; Protein Domains; RNA; Shelterin Complex; Substrate Specificity; Telomere; Templates, Genetic
PubMed: 35830881
DOI: 10.1038/s41586-022-05040-1