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Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR

February 13, 2022 Lieven Leave a comment

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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An open source protein gel documentation system for proteome analyses

February 13, 2022 Lieven Leave a comment

Data organization and data mining represents one of the main challenges for modern high throughput technologies in pharmaceutical chemistry and medical chemistry. The presented open source documentation and analysis system provides an integrated solution (tutorial, setup protocol, sources, executables) aimed at substituting the traditionally used lab-book.

The data management solution provided incorporates detailed information about the processing of the gels and the experimental conditions used and includes basic data analysis facilities which can be easily extended.

Cost-effective gel documentation using a web-cam.

In search for a cost effective gel documentation system applicable for different fields of molecular biology, we analyzed the capabilities of a cheap CCD-camera originally designed to capture images for transmission through the internet (web-cam) with regard to gel documentation. The camera was connected to a personal computer https://biodas.org/ by universal serial bus (USB) and used for the documentation of DNA separated on agarose gels and stained by ethidium-bromide using the software provided with the camera.

The web-cam provided digital images of sufficient quality for routine documentation and combined the low set-up costs of a Polaroid system with the low running costs of video capture systems, hence is ideal as a start-up system and as augmentation to existing equipment.

Persistence of the same Candida albicans strain despite fluconazole therapy. Documentation by pulsed-field gel electrophoresis.

Candida albicans and other Candida species have emerged as major nosocomial pathogens associated with a high mortality. Therapeutic options for fungal infections are limited. Amphotericin B has been the mainstay of treatment for serious systemic candidal infections, but it is relatively toxic and associated with a variety of side effects. Fluconazole has been proposed as alternative therapy for the treatment of systemic candidiasis including candidemia. We report the case of a patient with fungemia in whom fluconazole failed to eradicate C. albicans and C. tropicalis.

These pathogens were recovered from sputum and urine cultures, respectively, on day 12 of intravenous fluconazole therapy. Molecular epidemiologic techniques employing pulsed-field gel electrophoresis confirmed the persistence of the same C. albicans strain. Susceptibility studies showed a marked change in MICs of fluconazole between 24 and 48 hr, with an increase from less than or equal to 1.25 to greater than 80 micrograms/ml. Controlled trials will be needed to delineate the role of fluconazole in the treatment of disseminated candidiasis and its efficacy in comparison with amphotericin B. Amphotericin B should remain the drug of choice for such infections until data from controlled trials are available.

Inhibition of post-surgery tumour recurrence via a sprayable chemo-immunotherapy gel releasing PD-L1 antibody and platelet-derived small EVs

Background: Melanoma is the most serious type of skin cancer, and surgery is an effective method to treat melanoma. Unfortunately, local residual micro-infiltrated tumour cells and systemic circulating tumour cells (CTCs) are significant causes of treatment failure, leading to tumour recurrence and metastasis.

Methods: Small EVs were isolated from platelets by differential centrifugation, and doxorubicin-loaded small EVs (PexD) was prepared by mixing small EVs with doxorubicin (DOX). PexD and an anti-PD-L1 monoclonal antibody (aPD-L1) were co-encapsulated in fibrin gel. The synergistic antitumour efficacy of the gel containing PexD and aPD-L1 was assessed both in vitro and in vivo.

Results: Herein, we developed an in situ-formed bioresponsive gel combined with chemoimmunotherapeutic agents as a drug reservoir that could effectively inhibit both local tumour recurrence and tumour metastasis. In comparison with a DOX solution, PexD could better bind to tumour cells, induce more tumour immunogenic cell death (ICD) and promote a stronger antitumour immune response. PexD could enter the blood circulation through damaged blood vessels to track and eliminate CTCs. The concurrent release of aPD-L1 at the tumour site could impair the PD-1/PD-L1 pathway and restore the tumour-killing effect of cytotoxic T cells. This chemoimmunotherapeutic strategy triggered relatively strong T cell immune responses, significantly improving the tumour immune microenvironment.

Conclusion: Our findings indicated that the immunotherapeutic fibrin gel could “awaken” the host innate immune system to inhibit both local tumour recurrence post-surgery and metastatic potential, thus, it could serve as a promising approach to prevent tumour recurrence.

Clinical Comparison of I-gel and Laryngeal Mask Airway-Supreme Airway Devices During General Anaesthesia in the Paediatric Population

Objectives: Both the Supreme Laryngeal Mask Airway (SLMA) and the I-gel (I-gel) are supraglottic airway devices (SADs) commonly used for airway management in paediatric patients. This study aims to compare the efficacy in terms of insertion and ventilation profiles of size 2 SLMA and the I-gel in anaesthetised paediatric patients.

Methods: 100 children were prospectively allocated to two groups depending upon the device inserted as SLMA (n = 50) and I-gel (n = 50). The primary outcomes were studied in terms of ease of insertion, haemodynamic changes, ventilation parameters, leak pressure and incidences of complications during general anaesthesia.

Results: There were no failed attempts in the insertion of the airways in either group. The SLMA was more easily inserted in the majority of cases compared to the I-gel group. The number of attempts for insertion and the time taken for insertion were comparable in the I-gel and the SLMA group (13.8462.38 vs. 14.0261.7) (P .57, .66). Securing an effective airway took <30 seconds in both the groups with an overall median duration of 15 seconds. There was no difficulty in passing the gastric tube in either group (P<.30). There was a statistical difference between the oropharyngeal seal pressure (OSP), which was 25.1861.59 and 22.1061.36 cmH2O for SLMA and I-gel, respectively (P<.001). Haemodynamic parameters after the insertion of the device were comparable, and there were no clinically important complications in the post-operative period.

Conclusions: Both the devices appeared to be simple and suitable for airway management during elective surgery in paediatric patients. However, the SLMA was easily inserted with less insertion time in the majority of patients. Also, it provides higher OSP during anaesthesia and is better tolerated during emergence, with minimal risk of injury to the oropharynx.

Transfer and Fixation of Denatured RNA in Agarose Gels to Membranes by Capillary Transfer

In most cases, fractionation of RNA by agarose gel electrophoresis is but a prelude to hybridization of the fractionated population to specific labeled probes that detect particular target mRNAs. RNA is first transferred from an agarose gel to a 2D support, usually a nylon membrane.

This protocol presents the steps involved in the transfer of RNA from an agarose gel to a membranous support, facilitated by the upward flow of buffer, followed by various methods for fixation of the RNA to the membrane in preparation for hybridization. An alternative method for transfer by downward capillary flow is also given.

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