Validation of the Pockit Dengue Virus Reagent Set for Rapid Detection of Dengue Virus in Human Serum on a Field-Deployable PCR System.
Dengue virus (DENV) infection, a mosquito-borne disease, is a major public health problem in tropical countries. Point-of-care DENV detection with good sensitivity and specificity Bio Med Frontiers enables timely early diagnosis of DENV infection, facilitating effective disease management and control, particularly in regions of low resources.
The Pockit dengue virus reagent set (GeneReach Biotech), a reverse transcription insulated isothermal PCR (RT-iiPCR), is available to detect all four serotypes of DENV on the field-deployable Pockit system, which is ready for on-site applications. In this study, the analytical and clinical performances of the assay were evaluated.
The index assay did not react with 14 non-DENV human viruses, indicating good specificity.
Compared to the U.S. CDC DENV-1-4 real-time quantitative RT-PCR (qRT-PCR) assay, testing with serial dilutions of virus-spiked human sera demonstrated that the index assay had detection endpoints that were separately comparable with the 4 serotypes.
Excellent reproducibility was observed among repeat tests done by six operators at three sites.
In clinical performance, 195 clinical sera collected around Kaohsiung city in 2012 and 21 DENV-4-spiked sera were tested with the RT-iiPCR and qRT-PCR assays in parallel. The 121 (11 DENV-1, 78 DENV-2, 11 DENV-3, and 21 DENV-4) qRT-PCR-positive and 95 qRT-PCR-negative samples were all positive and negative by the RT-iiPCR reagent results, respectively, demonstrating high (100%) interrater agreement (95% confidence interval [CI95%], ∼98.81% to 100%; κ = 1).
With analytical and clinical performance equivalent to those of the reference qRT-PCR assay, the index PCR assay on the field-deployable system can serve as a highly sensitive and specific on-site tool for DENV detection.
Hapten-Functionalized DNA-Streptavidin Nanocircles as Supramolecular Reagents in a Competitive Immuno-PCR Assay.
Analysis with nanorings: The endogeneous proteins of supramolecular DNA nanocircles, obtained in high yields from oligomeric precursors containing bisbiotinylated DNA and streptavidin, are conveniently functionalized with biotinylated hapten moieties.
Our Provider These modular conjugates can be used as reagents in a novel competitive immunoassay for the high-sensitivity detection of low molecular weight analytes.
Evaluation of Direct PCR Amplification Using Various Swabs and Washing Reagents.
DNA profiles were generated via direct amplification from blood and saliva samples deposited on various types of swab substrates.
Each of the six non-FTA substrates used in this research was punched with a Harris 1.2 mm puncher. After 0.1 μL of blood or 0.5 μL saliva, samples were deposited on each of these punches, samples were pretreated with one of four buffers and washing reagents.
Amplification was performed using direct and nondirect autosomal and Y-STR kits. Autosomal and Y-STR profiles were successfully generated from most of these substrates when pretreated with buffer or washing reagents. Concordant profiles were obtained within and between the six substrates, the six amplification kits, and all four reagents.
The direct amplification of substrates which do not contain lysing agent would be beneficial to the forensic community as the procedure can be used on evidence samples commonly found at crime scenes.
Comparative Performance of Reagents and Platforms for Quantitation of Cytomegalovirus DNA by Digital PCR.
A potential benefit of digital PCR is a reduction in result variability across assays and platforms. Three sets of PCR reagents were tested on two digital PCR systems (Bio-Rad and RainDance), using three different sets of PCR reagents for quantitation of cytomegalovirus (CMV).
Both commercial quantitative viral standards and 16 patient samples (n = 16) were tested. Quantitative accuracy (compared to nominal values) and variability were determined based on viral standard testing results.
Quantitative correlation and variability were assessed with pairwise comparisons across all reagent-platform combinations for clinical plasma sample results.
The three reagent sets, when used to assay quantitative standards on the Bio-Rad system, all showed a high degree of accuracy, low variability, and close agreement with one another.
When used on the RainDance system, one of the three reagent sets appeared to have a much better correlation to nominal values than did the other two.
Quantitative results for patient samples showed good correlation in most pairwise comparisons, with some showing poorer correlations when testing samples with low viral loads.
Digital PCR is a robust method for measuring CMV viral load. Some degree of result variation may be seen, depending on platform and reagents used; this variation appears to be greater in samples with low viral load values.
Highly Selective Detection of 5-Methylcytosine in Genomic DNA Based on Asymmetric PCR and Specific DNA Damaging Reagents.
- DNA methylation is a significant epigenetic modification of the genome that is involved in regulating many cellular processes.
- An increasing number of human diseases have been discovered to be associated with aberrant DNA methylation, and aberrant DNA methylation has been deemed to be a potential biomarker for diseases such as cancers.
- A safe, nontoxic, and sensitive method for accurate detection of 5-methylcytosine in genomic DNA is extremely useful for early diagnosis and therapy of cancers.
- In this paper, we established a novel system to detect 5-methylcytosine, which is based on bisulfite treatment, asymmetric PCR, and specific DNA damaging reagents.
- Our method could be used for identifying the loci of 5mC in genomic DNA and detecting the DNA methylation levels in tissues as well.
Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination.
- Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods.
- However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays.
- Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process.
- Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions.
- These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase.
- Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents.
- On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants.
- As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of false-positive results in highly sensitive molecular fungal detection assays.
Ready to use dry-reagent PCR assays for the four common bacterial pathogens using a switchable lanthanide luminescence probe system.
Ready to use dry-reagent PCR assays for Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas spp. and for broad-range bacteria detection were developed. The assays were based on novel switchable lanthanide probes that provide sensitive target DNA detection with an exceptionally high signal-to-background ratio, thus enabling clear discrimination between positive and negative results.
Magnesium Chloride 25mM, PCR Reagent |
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11170057-1 | Glycomatrix | 10 mL | 13 EUR |
Magnesium Chloride 25mM, PCR Reagent |
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11170057-2 | Glycomatrix | 25 mL | 21.41 EUR |
Magnesium Chloride 25mM, PCR Reagent |
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11170057-3 | Glycomatrix | 50 mL | 34.4 EUR |
EP Reagent Biuret Reagent - 100ML |
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1011601 | Scientific Laboratory Supplies | 1L | 59.4 EUR |
PEROXIDE BLOCKING REAGENT Reagent |
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GWB-Q00308 | GenWay Biotech | 50 ml | Ask for price |
EP Reagent Iodoplatinate Reagent - 200ML |
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1046300 | Scientific Laboratory Supplies | 200ML | 502.2 EUR |
EP Reagent Molybdovanadic Reagent - 100ML |
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1056700 | Scientific Laboratory Supplies | 100ML | 59.4 EUR |
EP Reagent Methoxyphenylacetic Reagent - 100ML |
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1053601 | Scientific Laboratory Supplies | 100ML | 490.05 EUR |
EP Reagent Phosphomolybdotungstic Reagent - 100ML |
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1065000 | Scientific Laboratory Supplies | 100ML | 226.8 EUR |
AVIDIN/BIOTIN BLOCKING REAGENT Reagent |
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GWB-Q00289 | GenWay Biotech | 30 ml | Ask for price |
Gordon-McLeod Reagent (Oxidase reagent) |
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R026-100ML | EWC Diagnostics | 1 unit | 7.76 EUR |
Oxidase Reagent (Gordon-Macleod Reagent) |
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76207 | Sisco Laboratories | 100 ml | 3.38 EUR |
EP Reagent Sulfomolybdic Reagent R3 - 1L |
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1086500 | Scientific Laboratory Supplies | 1L | 398.25 EUR |
TRIpure Reagent (same as Trizol Reagent) |
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MBS356001-INQUIRE | MyBiosource | INQUIRE | Ask for price |
For example, sensitivity of three S. aureus and two S. pneumonia bacteria (colony forming units) per PCR assay was measured with fluorescence signal more than 30 times over the background signal level. The rapid and easy-to-use assays are suitable for routine clinical diagnostics without molecular biology expertise and facilities.