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Journal of Helminthology Nov 2023Molecular techniques are an alternative for the diagnosis of strongyloidiasis, produced by However, it is necessary to determine the best amplification target for the...
Molecular techniques are an alternative for the diagnosis of strongyloidiasis, produced by However, it is necessary to determine the best amplification target for the populations of this parasite present in a geographical area and standardize a polymerase chain reaction (PCR) protocol for its detection. The objectives of this work were the comparison of different PCR targets for molecular detection of and the standardization of a PCR protocol for the selected target with the best diagnostic results. DNA extraction was performed from parasite larvae by saline precipitation. Three amplification targets of the genes encoding ribosomal RNA 18S (18S rDNA) and 5.8S (5.8S rDNA) and cytochrome oxidase 1 (COX1) of were compared, and the PCR reaction conditions for the best target were standardized (concentration of reagents and template DNA, hybridization temperature, and number of cycles). The analytical sensitivity and specificity of the technique were determined. DNA extraction by saline precipitation made it possible to obtain DNA of high purity and integrity. The ideal target was the 5.8S rDNA, since the 18S rDNA yielded non-reproducible results and COX1 never amplified under any condition tested. The optimal conditions for the 5.8S rDNA-PCR were: 1.5 mM MgCl, 100 μM dNTPs, 0.4 μM primers, and 0.75 U DNA polymerase, using 35 cycles and a hybridization temperature of 60 °C. The analytical sensitivity of the PCR was 1 attogram of DNA, and the specificity was 100%. Consequently, the 5.8S rDNA was shown to be highly sensitive and specific for the detection of DNA.
Topics: Animals; Strongyloides stercoralis; Strongyloidiasis; Polymerase Chain Reaction; RNA, Ribosomal, 18S; DNA, Ribosomal; Feces
PubMed: 37974436
DOI: 10.1017/S0022149X23000743 -
Poultry Science Oct 2023Essential oils (EO) and natural bioactive compounds are well-known antibacterial and anti-inflammatory factors; however, little is known about their anticoccidial...
Essential oils (EO) and natural bioactive compounds are well-known antibacterial and anti-inflammatory factors; however, little is known about their anticoccidial activity and mode of action. EO deriving from basil (BEO), garlic (GAR), oregano (OEO), thyme (TEO), and their main bioactive compounds were investigated for their anticoccidial proprieties and compared to salinomycin (SAL) and amprolium (AMP) in vitro. The invasion of Eimeria tenella sporozoites was studied on 2 cell models: Madin-Darby Bovine Kidney (MDBK) cells and primary chicken epithelial cells (cIEC). Invasion efficiency was evaluated at 2 and 24 h postinfection (hpi) with counts of extracellular sporozoites and by detection of intracellular E. tenella DNA by PCR. Results show that at both timepoints, the EO were most effective in preventing the invasion of E. tenella with an average reduction of invasion at 24 hpi by 36% in cIEC and 55% in MDBK. The study also examined cytokine gene expression in cIEC at 24 hpi and found that AMP, BEO, OEO, TEO, carvacrol (CAR), and thymol (THY) significantly reduced interleukin (IL)8 expression, with CAR also reducing expression of IL1β and IL6 compared to the infected control. In addition, this work investigated the morphology of E. tenella sporozoites treated with anticoccidial drugs and EO using a scanning electron microscope. All the treatments induced morphological anomalies, characterized by a reduction of area, perimeter and length of sporozoites. SAL had a significant impact on altering sporozoite shape only at 24 h, whereas CAR and THY significantly compromised the morphology already at 2 hpi, compared to the untreated control. OEO and GAR showed the most significant alterations among all the treatments. The findings of this study highlight the potential of EO as an alternative to traditional anticoccidial drugs in controlling E. tenella invasion and in modulating primary immune response.
Topics: Animals; Cattle; Eimeria tenella; Oils, Volatile; Chickens; Sporozoites; Polymerase Chain Reaction; Coccidiosis; Cattle Diseases
PubMed: 37573847
DOI: 10.1016/j.psj.2023.102898 -
Scientific Reports Nov 2023Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2-3...
Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2-3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50-60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.
Topics: Animals; Humans; RNA; Polymerase Chain Reaction; RNA, Messenger; Cell Line; Biological Assay; Real-Time Polymerase Chain Reaction; Mammals
PubMed: 37923834
DOI: 10.1038/s41598-023-46314-6 -
Mycoses Dec 2023Onychomycosis is the most common nail pathology, involving various pathogens such as dermatophytes, moulds and yeasts.
BACKGROUND
Onychomycosis is the most common nail pathology, involving various pathogens such as dermatophytes, moulds and yeasts.
OBJECTIVE
The objective of this study was to observe the prevalence of onychomycosis, analyse the most appropriate diagnostic test, and assess the distribution of pathogens based on age, sex, quarter of the year, duration of symptoms and previous treatment.
METHODS
Retrospectively, mycological culture and PCR data and results were collected from 121 patients.
RESULTS
Of the 121 samples, 57% (69/121) tested positive when both microbiological study techniques were combined. The prevalence of onychomycosis was higher when PCR was performed (52.1%) compared to microbiological culture (33.1%). Among the 81 samples negative by microbiological culture, 31 were positive by PCR. Similarly, of the 58 samples negative by PCR, eight were positive by microbiological culture. Diagnostic accuracy data (with 95% confidence intervals) for PCR, using microbiological culture as the gold standard, were as follows: sensitivity of 0.8, specificity of 0.62, positive predictive value of 0.51 and negative predictive value of 0.86. The most frequently identified pathogen was Trichophyton rubrum, and the hallux nail plate was the most commonly affected location. However, no statistically significant associations were found between sex, age, quarter of the year and affected area with culture and PCR results.
CONCLUSION
Combining microbiological culture and PCR can increase the detection rate of onychomycosis and help avoid false-negative results.
Topics: Humans; Onychomycosis; Retrospective Studies; Sensitivity and Specificity; DNA, Fungal; Polymerase Chain Reaction
PubMed: 37574461
DOI: 10.1111/myc.13648 -
Journal of Advanced Research Aug 2023Early detection of pathogen-associated diseases are critical for effective treatment. Rapid, specific, sensitive, and cost-effective diagnostic technologies continue to... (Review)
Review
BACKGROUND
Early detection of pathogen-associated diseases are critical for effective treatment. Rapid, specific, sensitive, and cost-effective diagnostic technologies continue to be challenging to develop. The current gold standard for pathogen detection, polymerase chain reaction technology, has limitations such as long operational cycles, high cost, and high technician and instrumentation requirements.
AIM OF REVIEW
This review examines and highlights the technical advancements of CRISPR-Cas in pathogen detection and provides an outlook for future development, multi-application scenarios, and clinical translation.
KEY SCIENTIFIC CONCEPTS OF REVIEW
Approaches enabling clinical detection of pathogen nucleic acids that are highly sensitive, specific, cheap, and portable are necessary. CRISPR-Cas9 specificity in targeting nucleic acids and "collateral cleavage" activity of CRISPR-Cas12/Cas13/Cas14 show significant promise in nucleic acid detection technology. These methods have a high specificity, versatility, and rapid detection cycle. In this paper, CRISPR-Cas-based detection methods are discussed in depth. Although CRISPR-Cas-mediated pathogen diagnostic solutions face challenges, their powerful capabilities will pave the way for ideal diagnostic tools.
Topics: CRISPR-Cas Systems; Nucleic Acids; Polymerase Chain Reaction; Technology; Translations
PubMed: 36367481
DOI: 10.1016/j.jare.2022.10.011 -
Letters in Applied Microbiology Aug 2023A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human...
A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human infections. In this study, a polymerase chain reaction (PCR) protocol was designed to detect the pESI plasmid and confirm the Infantis serotype of Salmonella isolates. Primers were tested bioinformatically to predict specificity, sensitivity, and precision. A total of 54 isolates of Salmonella serotypes Infantis, Senftenberg, and Alachua were tested, with and without the pESI plasmid carriage. Isolates of 31 additional serotypes were also screened to confirm specificity to Infantis. Specificity, sensitivity, and precision of each primer were >0.95. All isolates tested produced the expected band sizes. This PCR protocol provides a rapid and clear result for the detection of the pESI plasmid and serotype Infantis and will allow for the in vitro detection for epidemiological studies where whole-genome sequencing is not available.
Topics: Animals; Humans; Salmonella; Plasmids; Salmonella enterica; Polymerase Chain Reaction; Disease Outbreaks
PubMed: 37505450
DOI: 10.1093/lambio/ovad088 -
Bulletin of Mathematical Biology Aug 2023We present a new approach for relating nucleic-acid content to fluorescence in a real-time Polymerase Chain Reaction (PCR) assay. By coupling a two-type branching...
We present a new approach for relating nucleic-acid content to fluorescence in a real-time Polymerase Chain Reaction (PCR) assay. By coupling a two-type branching process for PCR with a fluorescence analog of Beer's Law, the approach reduces bias and quantifies uncertainty in fluorescence. As the two-type branching process distinguishes between complementary strands of DNA, it allows for a stoichiometric description of reactions between fluorescent probes and DNA and can capture the initial conditions encountered in assays targeting RNA. Analysis of the expected copy-number identifies additional dynamics that occur at short times (or, equivalently, low cycle numbers), while investigation of the variance reveals the contributions from liquid volume transfer, imperfect amplification, and strand-specific amplification (i.e., if one strand is synthesized more efficiently than its complement). Linking the branching process to fluorescence by the Beer's Law analog allows for an a priori description of background fluorescence. It also enables uncertainty quantification (UQ) in fluorescence which, in turn, leads to analytical relationships between amplification efficiency (probability) and limit of detection. This work sets the stage for UQ-PCR, where both the input copy-number and its uncertainty are quantified from fluorescence kinetics.
Topics: Uncertainty; Mathematical Concepts; Models, Biological; Polymerase Chain Reaction; DNA
PubMed: 37574503
DOI: 10.1007/s11538-023-01182-z -
Nano Letters Feb 2024Rapid, sensitive, inexpensive point-of-care molecular diagnostics are crucial for the efficient control of spreading viral diseases and biosecurity of global health....
Rapid, sensitive, inexpensive point-of-care molecular diagnostics are crucial for the efficient control of spreading viral diseases and biosecurity of global health. However, the gold standard, polymerase chain reaction (PCR) is time-consuming and expensive and needs specialized testing laboratories. Here, we report a low-cost yet fast, selective, and sensitive Plasmonic Optical Wells-Based Enhanced Rate PCR: POWER-PCR. We optimized the efficient optofluidic design of 3D plasmonic optical wells via the computational simulation of light-to-heat conversion and thermophoretic convection in a self-created plasmonic cavity. The POWER-PCR chamber with a self-passivation layer can concentrate incident light to accumulate molecules, generate rapid heat transfer and thermophoretic flow, and minimize the quenching effect on the naked Au surface. Notably, we achieved swift photothermal cycling of nucleic acid amplification in POWER-PCR on-a-chip in 4 min 24 s. The POWER-PCR will provide an excellent solution for affordable and sensitive molecular diagnostics for precision medicine and preventive global healthcare.
Topics: Hot Temperature; Point-of-Care Testing; Computer Simulation; Polymerase Chain Reaction
PubMed: 38286020
DOI: 10.1021/acs.nanolett.3c04615 -
Expert Review of Molecular Diagnostics Mar 2024Molecular diagnostic systems for point-of-care (POC) testing are nowadays routinely used and are part of many labs. Although often intended for bedside use outside of... (Review)
Review
INTRODUCTION
Molecular diagnostic systems for point-of-care (POC) testing are nowadays routinely used and are part of many labs. Although often intended for bedside use outside of the microbiology lab, there is still room for expansion.
AREAS COVERED
This review discusses the two techniques that are currently the most widespread, real-time polymerase-chain reaction (RT-PCR) and loop-mediated isothermal amplification (LAMP). An overview is provided of the various manufacturers and products as well as the evidence and current use in clinical practice. The article further sheds light on some newer techniques, such as CRISPR-based diagnostics and lab-on-a-chip, which are still in development.
EXPERT OPINION
With many new platforms and techniques still in the pipeline and their potential currently not yet fully exploited, we expect the use of molecular POC testing to increase in the years to come. However, even when used in hospital - in lab, the main advantages of the tests being fast and easy to perform already provide significant benefits in terms of patient outcome.
Topics: Humans; Critical Illness; Point-of-Care Testing; Nucleic Acid Amplification Techniques; Molecular Diagnostic Techniques; Real-Time Polymerase Chain Reaction; Point-of-Care Systems; Sensitivity and Specificity
PubMed: 38414348
DOI: 10.1080/14737159.2024.2322146 -
Nature Biomedical Engineering Dec 2023The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the...
The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.
Topics: Humans; Mutation; Neoplasms; Polymerase Chain Reaction; DNA; Nucleotides
PubMed: 37500748
DOI: 10.1038/s41551-023-01072-8