Kit-based Methods Research Articles

On-site visual quantification of alkaline phosphatase activity in cells using a smartphone-based approach.

Alkaline phosphatase (ALP) is a critical biomarker associated with various physiological and pathological processes, making its detection essential for disease diagnosis and biomedical research. In this study, we developed a novel, simple, and portable visual quantification method for ALP activity in cells using an efficient Cu0.9Zn0.1S nanomaterial with peroxidase-like properties, integrated into a smartphone-based platform for enhanced usability. The Cu0.9Zn0.1S nanomaterial catalyzes the breakdown of H₂O₂, generating ·OH radicals that oxidize the colorless substrate TMB into blue oxTMB, which is subsequently reduced back to TMB by ascorbic acid (AA). We employed an indirect colorimetric approach for ALP detection, leveraging ALP's ability to catalyze the dephosphorylation of l-ascorbic acid-2-phosphate (AAP) to produce AA. This method enabled highly sensitive and precise quantification of ALP with a detection limit of 0.47 mU/L and a linear range from 0.001 to 100 U/L. The established smartphone platform not only facilitated real-time data processing but also transformed the detection system into a portable and accessible laboratory. The method was successfully applied to detect ALP in various cancer cell lines, with the highest levels found in HepG2 cells. Moreover, the results were consistent with those obtained using traditional kit-based methods, highlighting the accuracy and reliability of our approach. The system was also applied to evaluate ALP inhibitors, demonstrating its versatility in biomedical applications. This innovative, cost-effective, and highly sensitive colorimetric sensing strategy offers immense potential for clinical diagnostics, cancer research, and inhibitor screening, particularly in on-site, resource-limited, or emergency scenarios.

Using clotted, pelleted blood samples for direct molecular detection of Bartonella spp. in small mammal wildlife surveillance studies

ObjectiveBartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals.ResultsDNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.

Receptor-Targeted Peptide Conjugates Based on Diphosphines Enable Preparation of 99mTc and 188Re Theranostic Agents for Prostate Cancer.

Benchtop 99Mo/99mTc and 188W/188Re generators enable economical production of molecular theranostic 99mTc and 188Re radiopharmaceuticals, provided that simple, kit-based chemistry exists to radiolabel targeting vectors with these radionuclides. We have previously described a diphosphine platform that efficiently incorporates 99mTc into receptor-targeted peptides. Here, we report its application to label a prostate-specific membrane antigen (PSMA)-targeted peptide with 99mTc and 188Re for diagnostic imaging and systemic radiotherapy of prostate cancer. Methods: Two diphosphine-dipeptide bioconjugates, DP1-PSMAt and DP2-PSMAt, were formulated into kits for radiolabeling with 99mTc and 188Re. The resulting radiotracers were studied invitro, in prostate cancer cells, and invivo in mouse xenograft models, to assess similarity of uptake and biodistribution for each 99mTc/188Re pair of agents. Results: Both DP1-PSMAt and DP2-PSMAt could be efficiently radiolabeled with 99mTc and 188Re using kit-based methods to furnish the isostructural compounds M-DP1-PSMAt and M-DP2-PSMAt (M = [99mTc]Tc, [188Re]Re). All 99mTc/188Re radiotracers demonstrated specific uptake in PSMA-expressing prostate cancer cells, with negligible uptake in prostate cancer cells that did not express PSMA or in which PSMA uptake was blocked. M-DP1-PSMAt and M-DP2-PSMAt also exhibited high tumor uptake (18-30 percentage injected dose per gram at 2 h after injection), low retention in nontarget organs, fast blood clearance, and excretion predominantly via a renal pathway. Importantly, each pair of 99mTc/188Re radiotracers showed near-identical biologic behavior in these experiments. Conclusion: We have prepared and developed novel pairs of isostructural PSMA-targeting 99mTc/188Re theranostic agents. These generator-based theranostic agents have potential to provide access to the benefits of PSMA-targeted diagnostic imaging and systemic radiotherapy in health care settings that do not routinely have access to either reactor-produced 177Lu radiopharmaceuticals or PET/CT infrastructure.

Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa

Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used.

Mycoplasma bovis testing for the screening of semen imported into New Zealand

ABSTRACT Aims To evaluate the fitness of three PCR assays for the detection of Mycoplasma bovis in dilute (extended) bovine semen, and a reverse transcriptase-PCR (RT-PCR) adaptation as a proxy for viability. Materials and methods Four commercial kit-based methods for nucleic acid extraction were compared to test for the presence of PCR inhibitors in nucleic acid extracted from undiluted and diluted semen. Then, analytical sensitivity, analytical specificity, and diagnostic specificity of two real-time PCR and one conventional PCR were evaluated for the detection of M. bovis DNA in semen and compared against microbial culture. Furthermore, an RT-PCR was adapted to detect RNA only and tested on viable and non-viable M. bovis to establish its ability to discriminate between the two. Results No significant PCR inhibition was detected from the dilute semen. All DNA extraction methods except one were equivalent, regardless of semen dilution. The analytical sensitivity of the real-time PCR assays was estimated as 45.6 cfu per 200 µL semen straw (2.2 × 102 cfu/mL). The conventional PCR was 10 times less sensitive. No cross-reactivity was observed for the real-time PCR for any of the bacteria tested and the diagnostic specificity was estimated as 100 (95% CI = 94.04–100) %. The RT-PCR was poor in distinguishing between viable and non-viable M. bovis. The mean quantification cycle (Cq) values for RNA extracted from different treatments to kill M. bovis remained unchanged 0–48 hours after inactivation. Conclusion and clinical relevance The real-time PCR were fit for the purpose of screening dilute semen for the detection of M. bovis to prevent incursion via importation of infected semen. The real-time PCR assays can be used interchangeably. The RT-PCR could not reliably indicate the viability of M. bovis. Based on the results from this study, a protocol and guidelines have been produced for laboratories elsewhere that wish to test bovine semen for M. bovis.

Cost Effective Method for gDNA Isolation from the Cecal Content and High Yield Procedure for RNA Isolation from the Colonic Tissue of Mice.

Microbiome studies are quickly gaining momentum. Since most of the resident microbes (consisting of bacteria, fungi, and viruses) are difficult to culture, sequencing the microbial genome is the method of choice to characterize them. It is therefore important to have efficient methodology for gDNA isolation of gut microbes. Mouse models are widely used to understand human disease etiology while avoiding human ethics-related complications. However, the widely used kit-based methods are costly, and sometimes yields (in terms of quality and quantity) are sub-optimal. To overcome this problem, we developed a straightforward, standardized DNA isolation procedure from mouse cecal content for further microbiome-related studies. The reagents we used to standardize the procedure are readily available even in a not-so-well-equipped laboratory, and the reagents are not expensive. The yield and quality of the DNA are also better than those obtained by the readily available kit-based methods. Additionally, we modified the kit-based method of RNA isolation from the colon tissue sample of the mouse for better yield. Churning the tissue with liquid nitrogen at the beginning of the procedure improves RNA quality and quantity. Graphical abstract.

The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?

Historically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an "ready-to-use" (RTU) kit for steroids analysis.

Quality, quantity and recovery of DNA content from routine blood samples and genotyping success rate: comparison between phenol chloroform method (PCM) with a new kit-based DNA extraction method (KBM)

Background: DNA extraction has become a baseline method for molecular biology studies. There are a variety of methods available for this purpose. Newer kit-based methods (KBM) are easy and less time consuming than traditional chemical methods of extraction like phenol chloroform method (PCM). Though estimates of quality from different methods are available in labels, this study compared the practical outcomes regarding quantity, quality, DNA recovery rate and assessed the outcomes at two different time points.Methods: This study was done as a secondary analysis from an ongoing project. The quantity and quality of DNA isolated from the same group of 100 deidentified blood samples by PCM and KBM were analysed using Multi analyzer and repeated after a period of 3 months. Genotyping of the samples were done by RT-PCR. The quantity, quality and amplification proportion were compared between two groups to reach the inference.Results: The median (range) concentration of DNA by PCM was 543.27 (960.59) µg/ml and that of KBM was 32.115 (36.73) µg/ml. The quality of DNA as measured by absorbance at 260/280 nm was 1.84 in PCM and 1.81 in KBM (p>0.05). Genotyping success rate was 78% in PCM and 98% in KBM (p = 0.002). The DNA recovery rate was 96% in PCM and 80% in KBM (p=0.014).Conclusions: The median concentration of DNA obtained from PCM was more compared to KBM. The quality of DNA was comparable in both the groups. The genotyping success rate was more in KBM group. The DNA recovery rate at 3 months was more in PCM group.

Expression profiling of miR-96, miR-584 and miR-422a in colon cancer and their potential involvement in colon cancer pathogenesis

Purpose: To determine the correlation between miRNAs; miR-96, miR-422a and miR584, and colon cancer, and also to test whether any of these miRNAs can act as non-invasive biomarkers in colon cancerMethods: The tumor samples and the corresponding normal mucosa used in this study were collected from 60 patients diagnosed with colon cancer. HT-29, HCT-116 and SW-620 cell lines were used for miRNA expression profiling. Total RNA was extracted using kit-based methods with minor modifications, and specific miRNAs were detected and quantified by quantitative real-time polymerase chain reaction [RT-PCR].Results: The results indicate down-regulation of miR-96, miR-584 and miR-422a in colon cancer tissue and decreased expression levels in the three colon cancer cell lines (all p < 0.01). Lower miRNA expression levels are correlated with increased tumor size.Conclusion: This study shows that miR-96, miR-584 and miR-422a are down-regulated in colon cancer and are associated with tumor size. Thus, the ratio of miR-96/miR-638 in plasma is a potential noninvasive biomarker in colon cancer.Keywords: Colon cancer, miR-96, miR-422a, miR-584, Expression profiling, Biomarker

Extraction of high-quality genomic DNA from Ectocarpus.

For some applications, such as genome sequencing and high-throughput genotyping with multiple markers, it is necessary to use high-quality genomic DNA. This article describes how to obtain several micrograms of high-quality, cesium chloride-purified DNA from 1 g of Ectocarpus filaments. We also recommend using DNA of this quality for quantitative RT-PCR control reactions. However, simpler, more rapid, kit-based methods are preferable for experiments that involve the treatment of large numbers of individuals, such as genotyping large populations with a small number of markers or PCR screening of large populations.

Development and Testing of Methods to Screen Chickpea Flour for Starch Characteristics

AbstractRapid screening methodology has been developed for assessing the chemical composition and the gelatinisation and pasting properties of starch using flour from chickpea seeds. The methodology allows samples to be assessed using a minimal amount of sample and for starch information to be interpreted in the presence of other components present in the flour. The starch content of the flour and the amylose content of the starch was determined by modifying existing kit‐based methods (Megazyme International Ltd). Using the Differential Scanning Calorimeter (DSC), starch gelatinisation characteristics can be assessed based on temperature of gelatinisation (Tp), specific heat capacity (Cpsp ) and half width of transition (½ΔT). Pasting properties could be assessed using the Rapid Visco Analyser (RVA), based on the determination of onset temperature for the RVA profile (T \displaystyle _o^{RVA}), Tp − T \displaystyle _o^{RVA} and final viscosity (FV) values. The developed methodology was tested using a range of chickpea samples. No significant variation was found between the samples for starch content or for the proportion of amylose/amylopectin in the various starches. Significant variation was found in the starch properties of two of the samples, while the variation between the remaining samples was very small.