Development and validation of a cost-effective in-house method, tetra-primer ARMS PCR assay, in genotyping of seven clinically important point mutations

2011 ◽  
Vol 25 (4) ◽  
pp. 177-181 ◽  
Author(s):  
Ozdal Etlik ◽  
Vedat Koksal ◽  
S. Tugba Arican-Baris ◽  
Ibrahim Baris
Keyword(s):  
Point Mutations ◽  
Cost Effective ◽  
Pcr Assay ◽  
Arms Pcr ◽  
Development And Validation

scholarly journals A rapid and cost-effective multiplex ARMS-PCR method for the simultaneous genotyping of the circulating SARS-CoV-2 phylogenetic clades

2020 ◽  
Author(s):  
Md. Tanvir Islam ◽  
A. S. M. Rubayet Ul Alam ◽  
Najmuj Sakib ◽  
Md. Shazid Hasan ◽  
Tanay Chakrovarty ◽  
...  
Keyword(s):  
Low Income ◽  
High Throughput Sequencing ◽  
Cost Effective ◽  
Epidemiological Surveillance ◽  
Low Income Countries ◽  
Amplification Refractory Mutation System ◽  
Pcr Assay ◽  
Maximum Resolution ◽  
Arms Pcr ◽  
Mutation System

SummaryTracing the globally circulating SARS-CoV-2 mutants is essential for the outbreak alerts and far-reaching epidemiological surveillance. The available technique to identify the phylogenetic clades through high-throughput sequencing is costly, time-consuming, and labor-intensive that hinders the viral genotyping in low-income countries. Here, we propose a rapid, simple and cost-effective amplification-refractory mutation system (ARMS)-based multiplex reverse-transcriptase PCR assay to identify six distinct phylogenetic clades: S, L, V, G, GH, and GR. This approach is applied on 24 COVID-19 positive samples as confirmed by CDC approved real-time PCR assay for SARS-CoV-2. Our multiplex PCR is designed in a mutually exclusive way to identify V-S and G-GH-GR clade variants separately. The pentaplex assay included all five variants and the quadruplex comprised of the triplex variants alongside either V or S clade mutations that created two separate subsets. The procedure was optimized in the primer concentration (0.2-0.6 µM) and annealing temperature (56-60°C) of PCR using 3-5 ng/µl cDNA template synthesized upon random- and oligo(dT)-primer based reverse transcription. The different primer concentration for the triplex and quadruplex adjusted to different strengths ensured an even amplification with a maximum resolution of all targeted amplicons. The targeted Sanger sequencing further confirmed the presence of the clade-featured mutations with another set of our designed primers. This multiplex ARMS-PCR assay is sample, cost-effective, and convenient that can successfully discriminate the circulating phylogenetic clades of SARS-CoV-2.

scholarly journals Development and Validation of UV Spectrophotometric Method for the Determination of Cefuroxime Axetil in Bulk and Pharmaceutical Formulation

2013 ◽  
Vol 6 (1) ◽  
pp. 133-141 ◽  
Author(s):  
S. Binte Amir ◽  
M. A. Hossain ◽  
M. A. Mazid
Keyword(s):  
Spectrophotometric Method ◽  
Cost Effective ◽  
Cefuroxime Axetil ◽  
Pharmaceutical Formulation ◽  
Uv Spectrum ◽  
Accuracy And Precision ◽  
Relative Standard ◽  
Label Claim ◽  
Development And Validation

The present study was undertaken to develop and validate a simple, sensitive, accurate, precise and reproducible UV spectrophotometric method for cefuroxime axetil using methanol as solvent. In this method the simple UV spectrum of cefuroxime axetil in methanol was obtained which exhibits absorption maxima (?max) at 278 nm. The quantitative determination of the drug was carried out at 278 nm and Beer’s law was obeyed in the range of (0.80-3.60) µg/ml. The proposed method was applied to pharmaceutical formulation and percent amount of drug estimated (95.6% and 96%) was found in good agreement with the label claim. The developed method was successfully validated with respect to linearity, specificity, accuracy and precision. The method was shown linear in the mentioned concentrations having line equation y = 0.05x + 0.048 with correlation coefficient of 0.995. The recovery values for cefuroxime axetil ranged from 99.85-100.05. The relative standard deviation of six replicates of assay was less than 2%. The percent relative standard deviations of inter-day precision ranged between 1.45-1.92% and intra-day precision of cefuroxime axetil was 0.96-1.51%. Hence, proposed method was precise, accurate and cost effective.  Keywords: UV-Vis spectrophotometer; Method validation; Cefuroxime axetil; Recovery studies.  © 2013 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved.   doi: http://dx.doi.org/10.3329/jsr.v6i1.14879 J. Sci. Res. 6 (1), 133-141 (2013)  

scholarly journals A rapid and cost‐effective multiplex ARMS‐PCR method for the simultaneous genotyping of the circulating SARS‐CoV‐2 phylogenetic clades

2021 ◽  
Author(s):  
Mohammad Tanvir Islam ◽  
ASM Rubayet Ul Alam ◽  
Najmuj Sakib ◽  
Mohammad Shazid Hasan ◽  
Tanay Chakrovarty ◽  
...  
Keyword(s):  
Cost Effective ◽  
Arms Pcr ◽  
Pcr Method

Molecular Identification of Mutations Conferring Resistance to Azoxystrobin in Cercospora nicotianae

Plant Disease ◽  
2020 ◽  
Author(s):  
Hua Li ◽  
William Barlow ◽  
Ed Dixon ◽  
Bernadette F. Amsden ◽  
Robert Hirsch ◽  
...  
Keyword(s):  
Characteristic Curve ◽  
Point Mutations ◽  
Receiver Operator Characteristic Curve ◽  
Resistance Mutations ◽  
Pcr Assay ◽  
Cytb Sequence ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Tobacco Production ◽  
Moderately Resistant

Cercospora nicotianae, the causal agent of frogeye leaf spot (FLS) of tobacco, has been exposed to quinone outside inhibitor (QoI) fungicides for over a decade through azoxystrobin applications targeting other major foliar diseases. From 2016 to 2018, a total of 124 isolates were collected from tobacco fields throughout Kentucky. Sensitivity of these isolates to azoxystrobin was previously characterized by determining the effective concentration to inhibit 50% conidial germination (EC50). Based on azoxystrobin EC50, isolates were categorized into three discrete groups: high azoxystrobin sensitivity (< 0.08 µg/ml), moderate azoxystrobin sensitivity (0.14 to 0.64 µg/ml) and low azoxystrobin sensitivity (> 1.18 µg/ml). Variability in sensitivity in a limited number of C. nicotianae isolates was previously shown to be a result of resistance mutations in the fungicide target gene. To improve understanding of C. nicotianae cytochrome b (cytb) structure, the gene was cloned from three isolates representing each EC50group, and sequences were compared. Our analysis showed that cytb gene in C. nicotianae consists of 1161 nucleotides encoding 386 amino acids. Cytb sequence among the cloned isolates was identical with the exception of the F129L and G143A point mutations. To more rapidly determine the resistance status of C. nicotianae isolates to azoxystrobin, a PCR assay was developed to screen for mutations. Using this assay, 80% (n=99) of testedC. nicotianae isolates carried an F129L mutation and were moderately resistant to azoxystrobin, and 7% (n=9) carried the G143A mutation and were highly resistant. A receiver operator characteristic curve analysis suggested the PCR assay is a robust diagnostic tool to identify C. nicotianae isolates with different sensitivity to azoxystrobin in Kentucky tobacco production. The prevalence of both the F129L and G143A mutations in C. nicotianae from Kentucky differs from other pathosystems where resistance to QoI fungicides has been identified, in which the majority of sampled isolates of the pathogen species have a broadly-occurring cytb mutation.

scholarly journals Development and Validation of a Three-Dimensional Printed Multifunctional Trap for Surveillance of Mosquitoes

2021 ◽  
Vol 37 (2) ◽  
pp. 68-75
Author(s):  
Drew David Reinbold-Wasson ◽  
Michael Hay Reiskind
Keyword(s):  
Mosquito Species ◽  
Three Dimensional ◽  
Cost Effective ◽  
Disease Monitoring ◽  
Life History Stage ◽  
Mosquito Trap ◽  
Host Seeking ◽  
Vector Borne ◽  
Development And Validation ◽  
Better Than

ABSTRACT An essential component of vector-borne disease monitoring programs is mosquito surveillance. Surveillance efforts employ various collection traps depending on mosquito species and targeted life-history stage, i.e., eggs, larvae, host-seeking, resting, or gravid adults. Surveillance activities often use commercial traps, sometimes modified to accept specific mosquito species attractants. The advent of widely available and affordable 3D printing technology allows the construction of novel trap designs and components. The study goal was to develop and assess a cost-effective, multipurpose, 6-volt mosquito trap integrating features of both host-seeking and gravid mosquito traps to collect undamaged live specimens: a multifunctional mosquito trap (MMT). We tested the MMT in comparison to commercial traps, targeting gravid Aedes albopictus, host-seeking Ae. albopictus, and total number of host-seeking mosquitos regardless of species. Field evaluations found the MMT performed as well as or better than comparable commercial traps. This project demonstrates an easy to construct, inexpensive, and versatile mosquito trap, potentially useful for surveying multiple mosquito species and other hematophagous insects by varying attractants into the MMT.

scholarly journals Preclinical Validation of a Novel Injection-Molded Swab for the Molecular Assay Detection of SARS-CoV-2

Diagnostics ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 206
Author(s):  
Chiara E. Ghezzi ◽  
Devon R. Hartigan ◽  
Justin P. Hardick ◽  
Rebecca Gore ◽  
Miryam Adelfio ◽  
...  
Keyword(s):  
Health Care Providers ◽  
Medical Devices ◽  
Clinical Studies ◽  
Cost Effective ◽  
Care Providers ◽  
Nasal Tissue ◽  
Significant Difference ◽  
Injection Molded ◽  
Development And Validation

During the COVID-19 public health emergency, many actions have been undertaken to help ensure that patients and health care providers have timely and continued access to high-quality medical devices to respond effectively. The development and validation of new testing supplies and equipment, including collection swabs, has helped to expand the availability and capability for various diagnostic, therapeutic, and protective medical devices in high demand during the COVID-19 emergency. Here, we report the initial validation of a new injection-molded anterior nasal swab, ClearTip™, that was experimentally validated in a laboratory setting as well as in independent clinical studies in comparison to gold standard flocked swabs. We have also developed an in vitro anterior nasal tissue model which offers a novel, efficient, and clinically relevant validation tool to replicate the clinical swabbing workflow with high fidelity, while being accessible, safe, reproducible, and time- and cost-effective. ClearTip™ displayed greater inactivated virus release in the benchtop model, confirmed by its greater ability to report positive samples in a small clinical study in comparison to flocked swabs. We also quantified the detection of biological materials, as a proxy for viral material, in multi-center pre-clinical and clinical studies which showed a statistically significant difference in one study and a reduction in performance in comparison to flocked swabs. Taken together, these results emphasize the compelling benefits of non-absorbent injection-molded anterior nasal swabs for COVID-19 detection, comparable to standard flocked swabs. Injection-molded swabs, as ClearTip™, could have the potential to support future swab shortages, due to its manufacturing advantages, while offering benefits in comparison to highly absorbent swabs in terms of comfort, limited volume collection, and potential multiple usage.

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