Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR
Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations.
MSP requires multiple sample preparation steps including protein digestion, DNA isolation, and bisulfite conversion prior to detection. In this work, we present a streamlined assay platform and instrumentation for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples through the use of droplet magnetofluidic principles. In conjunction with this platform, we present a streamlined protocol for solid-phase https://biodas.org/ DNA extraction from cells and bisulfite conversion of genomic DNA, minimizing the processing steps and reagent volume for implementation on a compact assay platform.
Sensory analysis of hepatitis B virus DNA for medicinal clinical diagnostics based on molybdenum doped ZnO nanowires field effect transistor biosensor; a comparative study to PCR test results
In this paper, a bio-sensing setup for investigating hepatitis B virus deoxyribonucleic acid (HBV DNA) diagnosis including rapid testing and field effect transistor (FET) in label free assay is proposed. The FET biosensor was fabricated by molybdenum doped ZnO nanowires (NWs) in easy method and cost-free approach. The materialized NWs were synthesized by physical vapor deposition (PVD) growth mechanism.
The molybdenum dopant could bring about vacancy sites for DNA adsorption and electric charge transfer. The capability of the fabricated biosensor was evaluated by investigating the PCR-verified samples known as True Positive (TP), True Negative (TN), False Positive (FP) and False Negative (FN). The FET biosensor could materialize the clinical tests on samples TP, TN, FP and FN and could distinguish the clinical samples from each other. The designed biosensor showed more precision than the PCR-outcomes by exhibiting more sensitivity on labeled samples known as FN.
This research has analytical and comparative study on fabricated biosensor performance. The achieved results show that the biosensor had significant response to samples which have not been carefully detected by PCR test. The fabricated biosensor showed high accuracy, precision, sensitivity, specificity and reproducibility for differentiating (TP), (TN), (FP) and (FN) samples from healthy and normal sample. The response sensitivity was calculated and biosensor showed a detection limit (LOD) of 1 pM. The biosensor demonstrated high response and satisfied signal in the concentration ranges from 1 pM to 10 μM.
ACE2-based capacitance sensor for rapid native SARS-CoV-2 detection in biological fluids and its correlation with real-time PCR
- The spread of the SARS-CoV-2 and its increasing threat to human health worldwide have necessitated the development of new technological tools to combat the virus. Particular emphasis is given to the development of diagnostic methods that monitor the spread of the virus rapidly and effectively.
- In this study, we report the development and testing of an antibody-free biosensor, based on the immobilization of ACE2 protein on the surface of gold interdigitated electrode. When the sensor was used in laboratory conditions for targeting the virus’ structural spike protein, it showed a limit of detection [LOD] of 750 pg/μL/mm2. Thereafter, the response of the sensor to swab and saliva samples from hospitalized patients was examined.
- The virus presence in the samples was confirmed by electrical effective capacitance measurements executed on the biosensor, and correlated with real-time PCR results. We verified that the biosensor can distinguish samples that are positive for the virus from those that are negative in a total of 7 positive and 16 negative samples.
- In addition, the biosensor can be used for semi-quantitative measurement, since its measurements are divided into 3 areas, the negative samples, the weakly positive and the positive samples. Reproducibility of the experiments was demonstrated with at least 3 replicates and stability was tested by keeping the sensor standby for 7 days at 4 °C before repeating the experiment. This work presents a biosensor that can be used as a fast-screening test at point of care detection of SARS-CoV-2 since it needs less than 2 min to provide results and is of simple operation.
High-resolution melting PCR analysis for genotyping the gene polymorphism of TNF-α, TGF-β1, IL-10, and IFN-γ in lung transplant recipients
Background: High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation.
Objectives: This study evaluated HRM analysis as an option for detecting the single nucleotide polymorphism (SNP) of cytokine, and profiled the distribution of cytokine gene polymorphism in the lung transplant recipients (LTRs).
Material and methods: High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (-308 A/G), tumor growth factor beta 1 (TGF-β1) (+869 T/C), interleukin 10 (IL-10) (-592 C/A, -819 T/C, -1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) SNPs were developed on the LightCycler® 480. The SNPs of the aforementioned cytokine genes in 322 LTRs and 266 normal controls were detected using HRM-PCR approach. To confirm the accuracy of the HRM-PCR assay, we randomly selected 100 samples from the LTRs and detected the aforementioned SNPs with sequence-specific primer-polymerase chain reaction (SSP-PCR) method, using a commercial kit.
Results: The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0). Our data also show that the allele and genotype frequencies of the abovementioned cytokine are not significantly different between the LTRs and the control groups (p > 0.05). In addition, we found the genotypes of TGF-β1 +869 associated with high expression phenotype were prevalent in the LTRs. On the contrary, for TNF-α -308, IL-10 and IFN-γ, the genotypes associated with low expression phenotype were most common in the LTRs.
Conclusions: In this study, we described a rapid, low-cost and high-throughput HRM-PCR technology for genotyping cytokine SNPs. Our data may be utilized for future studies examining the associations of cytokine gene polymorphisms with the prognosis of the LTRs.
A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels
Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex.
To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR.
Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.
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