Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes

BackgroundDNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primers or probes regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed.MethodsWe calculate the hybridization temperatures of primer/probe sets after aligning to SARS-COV-2, SARS-COV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 °C to the fully matched reference temperature.ResultsWe obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes.ConclusionsSome primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.

Polyvalent Detection of Members of the Genus Potyvirus by Molecular Hybridization Using a Genus-Probe

The use of a unique riboprobe named polyprobe, carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the polyvalent detection of up to 10 different pathogens by using a nonradioactive molecular hybridization procedure. In the present analysis, we have developed a unique polyprobe with the capacity to detect all members of the genus Potyvirus, which we have named genus-probe. To do this, we have exploited the capacity of the molecular hybridization assay to cross-hybridize with related sequences by reducing the hybridization temperature. We observed that sequences showing a percentage similarity of 68% or higher could be detected with the same probe by hybridizing at 50 to 55°C, with a detection limit of picograms of viral RNA comparable to the specific individual probes. According to this, we developed several polyvalent polyprobes, containing three, five, or seven different 500-nucleotide fragments of a conserved region of the NIb gene. The polyprobe carrying seven different conserved regions was able to detect all the 32 potyviruses assayed in the present work with no signal in the healthy tissue, indicating the potential capacity of the polyprobe to detect all described, and probably uncharacterized, potyviruses being then considered as a genus-probe. The use of this technology in routine diagnosis not only for Potyvirus but also to other viral genera is discussed.

Phonon-assisted relaxation and decoherence of singlet-triplet qubits in Si/SiGe quantum dots

We study theoretically the phonon-induced relaxation and decoherence of spin states of two electrons in a lateral double quantum dot in a SiGe/Si/SiGe heterostructure. We consider two types of singlet-triplet spin qubits and calculate their relaxation and decoherence times, in particular as a function of level hybridization, temperature, magnetic field, spin orbit interaction, and detuning between the quantum dots, using Bloch-Redfield theory. We show that the magnetic field gradient, which is usually applied to operate the spin qubit, may reduce the relaxation time by more than an order of magnitude. Using this insight, we identify an optimal regime where the magnetic field gradient does not affect the relaxation time significantly, and we propose regimes of longest decay times. We take into account the effects of one-phonon and two-phonon processes and suggest how our theory can be tested experimentally. The spin lifetimes we find here for Si-based quantum dots are significantly longer than the ones reported for their GaAs counterparts.

Optimization and Application of Fluorescence in Situ Hybridization Assay for Detecting Polyphosphate - Accumulating Microorganisms

Laboratory-scale sequencing batch reactors (SBRs) were operated on activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Polyphosphate-accumulating microorganisms (PAOs) play an important role during the enhanced biological phosphorus removal (EBPR) process. Fluorescence in situ hybridization (FISH) was applied to assess the proportions of microorganisms in the sludge. The aim of this study was to optimize hybridization of PAOMIX and RHC439 probes by orthogonal design. Orthogonal optimization test of the four factors were conducted under the individual three levels. The optimal hybridizition conditions were as follow: hybridization temperature 46°C, hybridization time 2.5h, washing time 15min, formamide concentration 35%(PAOMIX probe); hybridization temperature 50°C, hybridization time 2.5h, washing time 20min, formamide concentration 20% (RHC439 probe).

Macroarray Detection of Solanaceous Plant Pathogens in the Fusarium solani Species Complex

Members of the Fusarium solani species complex (FSSC), which are morphologically similar but have more than 45 distinct lineages, were chosen as targets for the development of a macroarray detection system that would be broadly adaptable. Thirty-three oligonucleotides (17 to 27 mers) were designed from the internal transcribed spacer (ITS) of the ribosomal RNA genes of 17 FSSC isolates, which belong to 12 phylogenetically closely related species. Of the 33 oligonucleotides on the array, 21 were useful in discriminating all 12 species, some of which had only a single nucleotide difference among them. The high specificity of this method was achieved by optimizing the hybridization temperature and oligo probe length, which had a more substantial effect on the array performance than the melting temperature and the DNA G+C content (G/C%) of the probes. The array was validated by testing inoculated greenhouse samples and diseased field plant samples.

Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria

ABSTRACT We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.

Potentiometric Detection of DNA Molecules Hybridization Using Gene Field Effect Transistor and Intercalator

ABSTRACTWe propose a new concept of a gene field effect transistor (FET) for detection of allele specific oligonucleotide hybridization, which is in principle based on charge density change at the gate insulator. The electrical characteristics of the gene FET were found to shift after specific binding of biomolecules at the surface of the gate insulator. Allele specific oligonucleotide hybridization and reaction between double-stranded DNA and intercalator were successfully detected with gene FETs because they have intrinsic charges in an aqueous solution. Ability to discriminate single nucleotide polymorphism (SNP) was also examined using the gene FET. Our results show that control of hybridization temperature and utilization of intercalator lead to more precise SNP analysis using the gene FET.

Optimization of Fast-Fluorescence in situ Hybridization with Repetitive α-Satellite Probes

A rapid FISH (fluorescence in situ hybridization) technique (Fast-FISH) for quantitative microscopy has been recently introduced. For highly repetitive D N A probes the hybridization (renaturation) time and the number of necessary washing steps were reduced considerably by omitting formamide or equivalent denaturing chemical agents. Due to low stringency conditions major and minor binding sites of the probes used showed visible FISH signals well suited for quantitative image-microscopy. The discrimination of minor and major binding sites was possible by automated image-processing. Here, a further, quantitative optimization of the Fast-FISH technique is described that allows to clearly discriminate major and minor binding sites of a-satellite probes by an easy image classification parameter. With respect to the optimization it was necessary to verify two sensitive parameters (hybridization time and temperature) of the given rapid FISH protocol. A s examples the systematic optimization for the two probes D 12 Z 2 (major binding site on the centromere of chromosome 12) and D 8 Z 2 (major binding site on the centromere of chromosom e 8) are shown. The optimal hybridiza­tion conditions concerning rapidness and quality of chromosome morphology were obtained using a hybridization temperature of 70 °C and a hybridization time of 60 min. For these conditions major and minor binding sites were clearly discriminated by the intensity maxi­ mum Smax of the corresponding FISH-spots.