scholarly journals Development and Validation of a Multi-Locus PCR-HRM Method for Species Identification in Mytilus Genus with Food Authenticity Purposes

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1684
Author(s):  
Marianela Quintrel ◽  
Felipe Jilberto ◽  
Matías Sepúlveda ◽  
María Elisa Marín ◽  
David Véliz ◽  
...  
Keyword(s):  
Species Identification ◽  
Limit Of Detection ◽  
Perfect Agreement ◽  
Single Nucleotide ◽  
Internal Validation ◽  
Wine Vinegar ◽  
Seafood Industry ◽  
Fresh Frozen ◽  
Development And Validation

DNA-based methods using informative markers such as single nucleotide polymorphism (SNPs) are suitable for reliable species identification (SI) needed to enforce compliance with seafood labelling regulations (EU No.1379/2013). We developed a panel of 10 highly informative SNPs to be genotyped by PCR-High resolution melting (HRM) for SI in the Mytilus genus through in silico and in vitro stages. Its fitness for purpose and concordance were assessed by an internal validation process and by the transference to a second laboratory. The method was applicable to identify M. chilensis, M. edulis, M. galloprovincialis and M. trossulus mussels, fresh, frozen and canned with brine, oil and scallop sauce, but not in preserves containing acetic acid (wine vinegar) and tomato sauce. False-positive and negative rates were zero. Sensitivity, expressed as limit of detection (LOD), ranged between 5 and 8 ng/μL. The method was robust against small variations in DNA quality, annealing time and temperature, primer concentration, reaction volume and HRM kit. Reference materials and 220 samples were tested in an inter-laboratory assay obtaining an “almost perfect agreement” (κ = 0.925, p < 0.001). In conclusion, the method was suitable for the intended use and to be applied in the seafood industry.

scholarly journals DEVELOPMENT AND VALIDATION OF ULTRAVIOLET-SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF FEBUXOSTAT FOR CONDUCTING IN-VITRO QUALITY CONTROL TESTS IN BULK AND PHARMACEUTICAL DOSAGE FORMS

2018 ◽  
Vol 11 (9) ◽  
pp. 115
Author(s):  
Jaspreet Kaur ◽  
Daljit Kaur ◽  
Sukhmeet Singh
Keyword(s):  
Spectrophotometric Method ◽  
Limit Of Detection ◽  
Pharmaceutical Formulations ◽  
Dosage Forms ◽  
Pharmaceutical Dosage Forms ◽  
Relative Standard ◽  
Limit Of Quantitation ◽  
Development And Validation

Objective: A simple, accurate, and selective ultraviolet-spectrophotometric method has been developed for the estimation of febuxostat in the bulk and pharmaceutical dosage forms.Method: The method was developed and validated according to International Conference on Harmonization (ICH Q2 R1) guidelines. The developed method was validated statistically with respect to linearity, range, precision, accuracy, ruggedness, limit of detection (LOD), limit of quantitation (LOQ), and recovery. Specificity of the method was demonstrated by applying different stressed conditions to drug samples such as acid hydrolysis, alkaline hydrolysis, oxidative, photolytic, and thermal degradation.Results: The study was conducted using phosphate buffer pH 6.8 and λmax was found to be 312 nm. Standard plot having a concentration range of 1–10 μg/ml showed a good linear relationship with R2=0.999. The LOD and LOQ were found to be 0.118 μg/ml and 0.595 μg/ml, respectively. Recovery and percentage relative standard deviations were found to be 100.157±0.332% and <2%, respectively.Conclusion: Proposed method was successfully applicable to the pharmaceutical formulations containing febuxostat. Thus, the developed method is found to be simple, sensitive, accurate, precise, reproducible, and economical for the determination of febuxostat in pharmaceutical dosage forms.

scholarly journals Development and Validation of a Spectrophotometric Method for Quantification and Dissolution Studies of Glimepiride in Tablets

2012 ◽  
Vol 9 (2) ◽  
pp. 993-998
Author(s):  
Madhusudhanareddy Induri ◽  
Bhagavan Raju M. ◽  
Rajendra Prasad Y. ◽  
Pavankumar Reddy K.
Keyword(s):  
Quantitative Determination ◽  
Spectrophotometric Method ◽  
Analytical Method ◽  
Limit Of Detection ◽  
Pharmaceutical Formulations ◽  
Spectrophotometric Methods ◽  
Dissolution Studies ◽  
Limit Of Quantitation ◽  
Development And Validation

The objective of present study was to develop and validate an analytical method for quantitative determination and dissolution studies of glimepiride in tablets. The glimepiride shows absorption maxima at 225 nm and obeyed Beer's law in the range of 6.0 – 14.0 µg/mL. The limit of detection and limit of quantitation were 0.06, and 0.17 µg/mL respectively. Percentage recovery of glimepiride for the proposed method ranged from 99.32 to 100.98% indicating no interference of the tablet excipients. It was concluded that the proposed method is simple, easy to apply, economical and used as an alternative to the existing spectrophotometric and non-spectrophotometric methods for the routine analysis of glimepiride in pharmaceutical formulations andin vitrodissolution studies.

scholarly journals Development and Validation of RP-HPLC-PDA Method for the Analysis of Diclofenac Sodium in the In Vitro Transdermal Permeation Samples

2019 ◽  
Vol 9 (2) ◽  
pp. 212-216 ◽  
Author(s):  
Saisrianusha Valluru ◽  
Buchi N Nalluri
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Skin Permeation ◽  
Diclofenac Sodium ◽  
Hplc Method ◽  
Limit Of Quantification ◽  
Validation Parameters ◽  
In Vitro Skin Permeation ◽  
Development And Validation

A new analytical method using high-performance liquid chromatography coupled with photo diode array detection was developed and validated for the quantification of Diclofenac (DIC) from in vitro skin permeation samples. Analysis was performed using a Phenomenex C18 column (150 x 4.6mm, 5µm) with 10mM ammonium acetate: Acetonitrile (62:38% v/v) as the mobile phase in isocratic mode and eluents were monitored at 276nm. DIC was eluted at 3.1min and showed a good linearity in the concentration range of 0.2-3µg/mL with a correlation coefficient >0.999. The validation parameters, such as specificity, linearity, accuracy and limit of detection, limit of quantification, precision, robustness fulfilled the regulatory requirements. The developed HPLC method was successfully used for the analysis of DIC in samples obtained from transdermal diffusate samples.

Evaluation of invA Diversity among Salmonella Species Suggests Why Some Commercially Available Rapid Detection Kits May Fail To Detect Multiple Salmonella Subspecies and Species

2019 ◽  
Vol 82 (4) ◽  
pp. 710-717 ◽  
Author(s):  
ARIEL J. BUEHLER ◽  
MARTIN WIEDMANN ◽  
ZEINA KASSAIFY ◽  
RACHEL A. CHENG
Keyword(s):  
Foodborne Pathogens ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
False Negative ◽  
Detection Methods ◽  
Test Results ◽  
Salmonella Spp ◽  
Development And Validation ◽  
In Silico Analyses

ABSTRACTinvA is a common molecular target for Salmonella-specific detection methods and is recommended by the U.S. Food and Drug Administration Bacteriological Analytical Manual as a target for PCR confirmation of putative Salmonella isolates. Novel assays designed for the rapid detection of foodborne pathogens are often validated according to guidelines provided by validation schemes, such as the AOAC International or the International Organization for Standardization. However, these validation guidelines allow for flexibility in the validation study experimental design, which may inflate the assay's ability to detect foodborne pathogens, especially for foodborne pathogens such as Salmonella, exhibiting tremendous species diversity with &gt;2,600 confirmed serovars. This study was conducted to (i) describe the sequence diversity of invA, across a diverse set of Salmonella serovars and (ii) evaluate the ability of two commercially available, AOAC International–validated rapid detection assays to detect a diverse collection of Salmonella spp. strains. In silico analyses identified 362 of 2,058 nucleotide sites that were variable among invA sequences from a diverse collection, representing 86 unique serovars spanning all species and subspecies. Not surprisingly, the majority of variable sites (308 of 2,058) occurred in non–Salmonella enterica subsp. enterica strains, including Salmonella bongori and the other S. enterica subspecies. In vitro testing showed that both rapid detection assays, examined here, failed to detect all Salmonella strains at 1 log above the limit of detection, with assay A failing to detect S. enterica subsp. salamae, and assay B failing to detect S. bongori. Both strains were eventually detected at 100,000 times the limit of detection. Taken together, our study highlights the need to include non–subsp. S. enterica strains in the development and validation of rapid detection methods to limit false-negative test results.HIGHLIGHTS

scholarly journals Development and Validation of an Automated Zone Fluidics-Based Sensor for In Vitro Dissolution Studies of Captopril Using Total Error Concept

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 824
Author(s):  
Theano D. Karakosta ◽  
Paraskevas D. Tzanavaras ◽  
Constantinos K. Zacharis
Keyword(s):  
Limit Of Detection ◽  
Total Error ◽  
In Vitro Dissolution ◽  
Flow Conditions ◽  
Limit Of Quantification ◽  
Intermediate Precision ◽  
Tolerance Intervals ◽  
Dissolution Tests ◽  
Development And Validation

In the present research, a zone fluidics-based automated sensor for the analysis of captopril in in vitro dissolution samples is reported. Captopril is reacted under flow conditions with Ni(II) (10 mmol L−1) in alkaline medium (0.15% v/v NH3) to form a stable derivate, which is monitored spectrophotometrically at 340 nm. The chemical and instrumental parameters were carefully investigated and optimized. The validation of the developed method was performed in the range of 5 to 120% of the expected maximum concentration using the accuracy profiles as a graphical decision-making tool. The β-expectation tolerance intervals did not exceed the acceptance criteria of ±10%, which means that 95% of future results will be encompassed in the defined bias limits. The variation of the relative bias ranged between −2.3% and 3.5% and the RSD values for repeatability and intermediate precision were lower than 2.3% in all cases. The limit of detection (LOD), and the lower and the upper limit of quantification (LLOQ, ULOQ) were satisfactory and found to be 1%, 5% and 120% (corresponding to 0.6, 2.78 and 66.67 μg mL−1 in dissolution medium). The developed method was successfully applied for the analysis of captopril in dissolution tests of two commercially available batches.

scholarly journals Computational Design of a Molecularly Imprinted Polymer for the Biomonitoring of the Organophosphorous Metabolite Chlorferron

Biosensors ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 192
Author(s):  
Bakhtiyar Qader ◽  
Issam Hussain ◽  
Mark Baron ◽  
Rebeca Jiménez-Pérez ◽  
Guzmán Gil-Ramírez ◽  
...  
Keyword(s):  
Biological Samples ◽  
Limit Of Detection ◽  
Computational Design ◽  
Signal Change ◽  
Highly Sensitive ◽  
Limit Of Quantitation ◽  
Molecularly Imprinting Polymers ◽  
Mip Sensor ◽  
Parasitic Mites

Coumaphos is an organophosphorus compound used as insecticide and frequently used by beekeepers for the management of parasitic mites. The most important metabolite, chlorferron (CFN), has been identified in biological samples and foodstuff. The need to quickly identify the presence of typical metabolites, as an indication of interaction with coumaphos has driven the need to produce a highly sensitive electrochemical method for chlorferron analysis, based on molecularly imprinting polymers (MIP) technology. It showed irreversible behaviour with mixed diffusion/adsorption-controlled reactions at the electrode surface. A monoelectronic mechanism of reaction for oxidation has also been suggested. The linear range observed was from 0.158 to 75 µM. Median precision in terms of %RSD around 3% was also observed. For DPV, the limit of detection (LOD) and the limit of quantitation (LOQ) for the CFN-MIP were 0.158 µM and 0.48 µM, respectively. The obtained median % recovery was around 98%. The results were also validated to reference values obtained using GC-MS. Urine and human synthetic plasma spiked with CFN were used to demonstrate the usability of the method in biological samples, showing the potential for biomonitoring. The developed imprinted sensor showed maximum signal change less than 16.8% when related metabolites or pesticide were added to the mix, suggesting high selectivity of the MIP sensor toward CFN molecules. The results from in vitro metabolism of CMP analysed also demonstrates the potential for detection and quantification of CFN in environmental samples. The newly developed CFN-MIP sensor offers similar LoDs than chromatographic methods with shorter analysis time.

scholarly journals Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexia Bordigoni ◽  
Anne Motte ◽  
Hervé Tissot-Dupont ◽  
Philippe Colson ◽  
Christelle Desnues
Keyword(s):  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Papillomaviruses ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
Gene Encoding ◽  
Hpv Dna ◽  
E6 And E7 ◽  
High Risk Hpv ◽  
Development And Validation

AbstractHuman papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes (E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 101 for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

scholarly journals Somatic variants for seed and fruit set in grapevine

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Laura Costantini ◽  
Paula Moreno-Sanz ◽  
Chinedu Charles Nwafor ◽  
Silvia Lorenzi ◽  
Annarita Marrano ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Fruit Set ◽  
Climatic Factors ◽  
Embryo Sac ◽  
Wine Industry ◽  
Nucleotide Polymorphisms ◽  
Wine Quality ◽  
Single Nucleotide ◽  
Germplasm Collections

Abstract Background Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. Results We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data. Conclusions Our observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, the VvAGL11 mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.

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