March 5, 2022
Development of a dry-reagent-based nucleic acid-sensing platform by coupling thermostabilised LATE-PCR assay to an oligonucleotide-modified lateral flow biosensor.
- In this study, we report for the first time the development of a dry-reagent-based nucleic acid-sensing platform by combining a thermostabilised linear-after-the-exponential (LATE)-PCR assay with a one-step, Bio Med Frontiers hybridization-based nucleic acid lateral flow biosensor.
- The nucleic acid-sensing platform was designed to overcome the need for stringent temperature control during transportation or storage of reagents and reduces the dependency on skilled personnel by decreasing the overall assay complexity and hands-on time.
- The platform was developed using toxigenic Vibrio cholerae as the model organism due to the bacterium’s propensity to cause epidemic and pandemic cholera. The biosensor generates result which can be visualised with the naked eyes and the limit of detection was found to be 1pg of pure genomic DNA and 10CFU/ml of toxigenic V. cholerae.
- The dry-reagent-based nucleic acid-sensing platform was challenged with 95 toxigenic V. cholerae, 7 non-toxigenic V. cholerae and 66 other bacterial strains in spiked stool sample and complete agreement was observed when the results were compared to that of monosialoganglioside (GM1)-ELISA.
- Heat-stability of the thermostabilised LATE-PCR reaction mixes at different storage temperatures (4-56°C) was investigated for up to 90days. The dry-reagent-based genosensing platform with ready-to-use assay components provides an alternative method for sequence-specific detection of nucleic acid without any cold chain restriction that is associated with conventional molecular amplification techniques.
Adaption of SYBR Green-based reagent kit for real-time PCR quantitation of GC-rich DNA.
In the mammalian genome, approximately 50% of all genes are controlled by promoters with high GC contents. Analyzing the epigenetic mechanisms regulating their expression is difficult. Hence, we examined a method for stable quantification of such GC-rich DNA sequences.
Quantification of DNA during real-time PCR is often based on reagent kits containing the fluorescent dye SYBR Green. However, Our Provider these ready-made kits may not be suitable for amplifying DNA samples with a high GC content (>70%).
DNA segments with eccentric GC contents are frequently found in proximal promoter areas, and their quantification may be necessary in chromatin accessibility by real-time polymerase chain reaction or chromatin immunoprecipitation analyses of epigenetic mechanisms of gene regulation.
We therefore optimized the SYBR Green I FastStart reaction system by supplementing the system with dimethyl sulfoxide, betaine, and increased DNA polymerase content.
Here, we describe the development of the assay and demonstrate its effectiveness for two different DNA templates, showing that these modifications allow for the reliable amplification and quantification of DNA with GC contents exceeding >70% using the LightCycler instrument.
A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.
Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained.
For this purpose, we investigated two enhancer reagents, polyethylene glycol, and tRNA, for a chemical transfection method.
The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized.
The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3′-terminus of any gene.
The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.
Evaluation of cells and biological reagents for adventitious agents using degenerate primer PCR and massively parallel sequencing.
- We employed a massively parallel sequencing (MPS)-based approach to test reagents and model cell substrates including Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), African green monkey kidney (Vero), and High Five insect cell lines for adventitious agents.
- RNA and DNA were extracted either directly from the samples or from viral capsid-enriched preparations, and then subjected to MPS-based non-specific virus detection with degenerate oligonucleotide primer (DOP) PCR. MPS by 454, Illumina MiSeq, and Illumina HiSeq was compared on independent samples. Virus detection using these methods was reproducibly achieved. Unclassified sequences from CHO cells represented cellular sequences not yet submitted to the databases typically used for sequence identification.
- The sensitivity of MPS-based virus detection was consistent with theoretically expected limits based on dilution of virus in cellular nucleic acids. Capsid preparation increased the number of viral sequences detected. Potential viral sequences were detected in several samples; in each case, these sequences were either artifactual or (based on additional studies) shown not to be associated with replication-competent viruses.
- Virus-like sequences were more likely to be identified in BLAST searches using virus-specific databases that did not contain cellular sequences.
- Detected viral sequences included previously described retrovirus and retrovirus-like sequences in CHO, Vero, MDCK and High Five cells, and nodavirus and endogenous bracovirus sequences in High Five insect cells. Bovine viral diarrhea virus, bovine hokovirus, and porcine circovirus sequences were detected in some reagents.
- A recently described parvo-like virus present in some nucleic acid extraction resins was also identified in cells and extraction controls from some samples. The present study helps to illustrate the potential for MPS-based strategies in evaluating the presence of viral nucleic acids in various sample types, including cell culture substrates and vaccines.
Effects of a novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional stabilizing reagents.
Stabilization of nucleated blood cells by cell stabilizing reagent (BCT reagent) present in the Cell-Free DNA BCT blood collection device and consequent prevention of cell-free DNA contamination by cellular DNA during sample storage and shipping have previously been reported.
This study was conducted to investigate the effect of this novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional cell stabilizing reagents, formaldehyde and glutaraldehyde.
A 787 bp long DNA fragment from human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was amplified by PCR and used as model system.
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Enterovirus PCR kit |
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Enterovirus PCR kit |
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Mengovirus PCR kit |
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DNA samples and blood samples were treated with BCT reagent, 0.1% formaldehyde or 0.1% glutaraldehyde at room temperature.
DNA amplification was studied using conventional and real-time quantitative PCR.
Results indicate that exposure of DNA to the BCT reagent for up to 14 days had no effect on DNA amplification by PCR as compared to the untreated control DNA.
However, there was statistically significant decrease in DNA amplification in the DNA samples treated with formaldehyde and glutaraldehyde.
We conclude that the BCT reagent used in Cell-Free DNA BCT blood collection device to prevent cell-free DNA contamination by cellular DNA had no effect on DNA amplification by PCR.