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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 Microbiology Reviews Jan 1995DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of... (Review)
Review
DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified.
Topics: Animals; DNA Probes; Humans; Leishmaniasis; Malaria; Nematode Infections; Polymerase Chain Reaction; Protozoan Infections; Trypanosomiasis
PubMed: 7704890
DOI: 10.1128/CMR.8.1.113 -
Applied and Environmental Microbiology Sep 1991The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation....
The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed.
Topics: Acetyl Coenzyme A; Bacteria; DNA Probes; DNA, Bacterial; Formate-Tetrahydrofolate Ligase; Genetic Engineering; Manure; Nucleic Acid Hybridization
PubMed: 1768134
DOI: 10.1128/aem.57.9.2602-2609.1991 -
Genomics Jul 2017Polymerase chain reaction (PCR) is one of the most important laboratory techniques used in molecular biology, genetics and molecular diagnostics. The success of a...
Polymerase chain reaction (PCR) is one of the most important laboratory techniques used in molecular biology, genetics and molecular diagnostics. The success of a PCR-based method largely depends on the correct nucleic acid sequence analysis in silico prior to a wet-bench experiment. Here, we report the development of an online Java-based software for virtual PCR on linear or circular DNA templates and multiple primer or probe search from large or small databases. Primer or probe sensitivity and specificity are predicted by searching a database to find sequences with an optimal number of mismatches, similarity and stability. The software determines primer location, orientation, efficiency of binding and calculates primer melting temperatures for standard and degenerate oligonucleotides. The software is suitable for batch file processing, which is essential for automation when working with large amounts of data. The online Java software is available for download at http://primerdigital.com/tools/pcr.html. Accession numbers for the sequences resulting from this study: EU140956 EU177767 EU867815 EU882730 FJ975775-FJ975780 HM481419 HM481420 KC686837-KC686839 KM262797.
Topics: Computer Simulation; DNA Primers; DNA Probes; Polymerase Chain Reaction; Sequence Analysis, DNA; Software
PubMed: 28502701
DOI: 10.1016/j.ygeno.2017.05.005 -
BMC Infectious Diseases Apr 2006Due to an increasing number of norovirus infections in the last years rapid, specific, and sensitive diagnostic tools are needed. Reverse transcriptase-polymerase chain...
BACKGROUND
Due to an increasing number of norovirus infections in the last years rapid, specific, and sensitive diagnostic tools are needed. Reverse transcriptase-polymerase chain reactions (RT-PCR) have become the methods of choice. To minimize the working time and the risk of carryover contamination during the multi-step procedure of PCR the multiplex real-time RT-PCR for the simultaneous detection of genogroup I (GI) and II (GII) offers advantages for the handling of large amounts of clinical specimens.
METHODS
We have developed and evaluated a multiplex one-tube RT-PCR using a combination of optimized GI and GII specific primers located in the junction between ORF1 and ORF2 of the norovirus genome. For the detection of GI samples, a 3'-minor groove binder-DNA probe (GI-MGB-probe) were designed and used for the multiplex real-time PCR.
RESULTS
Comparable results to those of our in-house nested PCR and monoplex real-time-PCR were only obtained using the GI specific MGB-probe. The MGB-probe forms extremely stable duplexes with single-stranded DNA targets, which enabled us to design a shorter probe (length 15 nucleotides) hybridizing to a more conserved part of the GI sequences. 97% of 100 previously norovirus positive specimens (tested by nested PCR and/or monoplex real-time PCR) were detected by the multiplex real-time PCR. A broad dynamic range from 2 x 10(1) to 2 x 10(7) genomic equivalents per assay using plasmid DNA standards for GI and GII were obtained and viral loads between 2.5 x 10(2) and 2 x 10(12) copies per ml stool suspension were detected.
CONCLUSION
The one-tube multiplex RT real-time PCR using a minor groove binder-DNA probe for GI is a fast, specific, sensitive and cost-effective tool for the detection of norovirus infections in both mass outbreaks and sporadic cases and may have also applications in food and environmental testing.
Topics: Animals; Caliciviridae Infections; DNA Primers; DNA Probes; Gastroenteritis; Humans; Norovirus; RNA, Viral; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 16606447
DOI: 10.1186/1471-2334-6-69 -
Nucleic Acids Research Jul 2016We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method...
We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection.
Topics: Animals; DNA Ligases; DNA Probes; Humans; Mice; MicroRNAs; Nucleic Acid Hybridization; Rats; Real-Time Polymerase Chain Reaction; Viral Proteins
PubMed: 27154271
DOI: 10.1093/nar/gkw399 -
Cancer Genomics & Proteomics 2023Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors...
BACKGROUND/AIM
Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors on the localization of EGFR mutations in tumor tissues remain to be elucidated. Thus, a simple and efficient technology for the detection of mutations in tumor tissue specimens needs to be developed.
MATERIALS AND METHODS
Using an EGFR mutation-specific peptide nucleic acid (PNA)-DNA probe, the EGFR mutation-positive part of whole NSCLC tissues was visualized by immunofluorescence. Formalin-fixed paraffin-embedded sections obtained from A549, NCI-H1975, HCC827 and PC-9 tumors transplanted into nude mice were subjected to staining using PNA-DNA probes specific for the mRNA sequences producing the L858R, del E746-A750 and T790M mutations.
RESULTS
The probes for the L858R mutation showed intense positive staining in H1975 cells, and the probe for the del E746-A750 mutation exhibited positive staining specifically in HCC827 and PC-9 tumors. On the other hand, A549 tumors without EGFR mutation did not show any significant staining for any PNA-DNA probe. In combination staining, the addition of cytokeratin stain increased the positive staining rate of each PNA-DNA probe. In addition, the positive staining rate of the probes for the L858R mutation was comparable to that of the antibody to EGFR L858R mutated protein.
CONCLUSION
PNA-DNA probes specific for EGFR mutations might be useful tools to detect heterogeneous mutant EGFR expression in cancer tissues and efficiently evaluate the effect of EGFR signaling inhibitors on tissues of EGFR-mutant cancer.
Topics: Animals; Humans; Mice; Carcinoma, Non-Small-Cell Lung; DNA; DNA Probes; ErbB Receptors; Lung Neoplasms; Mice, Nude; Mutation; Peptide Nucleic Acids; Protein Kinase Inhibitors
PubMed: 37400147
DOI: 10.21873/cgp.20389 -
Genome Research Sep 1996Photoactivatable DNA analogs have been incorporated enzymatically into DNA and used to map the locations of polypeptides in protein complexes bound to DNA. We have...
Photoactivatable DNA analogs have been incorporated enzymatically into DNA and used to map the locations of polypeptides in protein complexes bound to DNA. We have developed a procedure for generating long primers from short oligodeoxyribonucleotides (oligos) to incorporate DNA cross-linkers at specific sites within either strand of DNA probes of < or = 206 bp. Single-stranded DNA molecules of 52-206 nucleotides in length were generated by asymmetric polymerase chain reactions (aPCR), using an excess of one short sense-strand primer to be extended and a limiting amount of each short antisense primer that is complementary to and defines the 3' end of the long primer to be generated. The noncross-linking strand of the DNA probe was also generated by aPCR from the DNA sequence of interest. The long primers were annealed to the full-length noncross-linking DNA strand to form a partially double-stranded DNA. Cross-linking analogs and radioactive deoxyribonucleotides (dNTPs), followed by normal dNTPs, were enzymatically incorporated onto the long primers to form the double-stranded DNA cross-linking probes. This method is reproducible and avoids many of the difficulties encountered by other published methods.
Topics: Base Sequence; Cross-Linking Reagents; DNA Primers; DNA Probes; DNA, Single-Stranded; Molecular Sequence Data; Polymerase Chain Reaction; Templates, Genetic; Transcription, Genetic
PubMed: 8889557
DOI: 10.1101/gr.6.9.886 -
Biosensors & Bioelectronics Jan 2017This paper presents a label-free affinity-based capacitive biosensor using interdigitated electrodes. Using an optimized process of DNA probe preparation to minimize the...
This paper presents a label-free affinity-based capacitive biosensor using interdigitated electrodes. Using an optimized process of DNA probe preparation to minimize the effect of contaminants in commercial thiolated DNA probe, the electrode surface was functionalized with the 24-nucleotide DNA probes based on the West Nile virus sequence (Kunjin strain). The biosensor has the ability to detect complementary DNA fragments with a detection limit down to 20 DNA target molecules (1.5aM range), making it suitable for a practical point-of-care (POC) platform for low target count clinical applications without the need for amplification. The reproducibility of the biosensor detection was improved with efficient covalent immobilization of purified single-stranded DNA probe oligomers on cleaned gold microelectrodes. In addition to the low detection limit, the biosensor showed a dynamic range of detection from 1µL to 10µL target molecules (20 to 2 million targets), making it suitable for sample analysis in a typical clinical application environment. The binding results presented in this paper were validated using fluorescent oligomers.
Topics: Base Sequence; Biosensing Techniques; DNA; DNA Probes; DNA, Single-Stranded; Electric Capacitance; Electrochemical Techniques; Electrodes; Equipment Design; Gold; Humans; Immobilized Nucleic Acids; Limit of Detection; Nucleic Acid Hybridization; Point-of-Care Systems; Reproducibility of Results; Sulfhydryl Compounds; West Nile Fever; West Nile virus
PubMed: 27619528
DOI: 10.1016/j.bios.2016.09.006 -
The Analyst Apr 2011Amplification-free detection of nucleic acids in complex biological samples is an important technology for clinical diagnostics, especially in the case where the...
Amplification-free detection of nucleic acids in complex biological samples is an important technology for clinical diagnostics, especially in the case where the detection is quantitative and highly sensitive. Here we present the detection of a synthetic DNA sequence from Herpes Simplex Virus-1 within swine cerebrospinal fluid (CSF), using a sandwich-like, magnetic nanoparticle pull-down assay. Magnetic nanoparticles and fluorescent polystyrene nanoparticles were both modified with DNA probes, able to hybridise either end of the target DNA, forming the sandwich-like complex which can be captured magnetically and detected by fluorescence. The concentration of the target DNA was determined by counting individual and aggregated fluorescent nanoparticles on a planar glass surface within a fluidic chamber. DNA probe coupling for both nanoparticles was optimized. Polystyrene reporter nanoparticles that had been modified with amine terminated DNA probes were also treated with amine terminated polyethylene glycol, in order to reduce non-specific aggregation and target independent adhesion to the magnetic particles. This way, a limit of detection for the target DNA of 0.8 pM and 1 pM could be achieved for hybridisation buffer and CSF respectively, corresponding to 0.072 and 0.090 femtomoles of target DNA, in a volume of 0.090 mL.
Topics: Animals; DNA Probes; DNA, Viral; Fluorescent Dyes; Magnetics; Nanoparticles; Nucleic Acid Hybridization; Polystyrenes; Simplexvirus; Spectrometry, Fluorescence; Swine
PubMed: 21369562
DOI: 10.1039/c0an01021a