Rapid Screening Method For Detection Research Articles

Evaluation of PCR technique as a rapid screening method for detection of group B streptococci colonization in pregnant women

Evaluation of PCR technique as a rapid screening method for detection of group B streptococci colonization in pregnant women

Evaluation of PCR technique as a rapid screening method for detection of Group B streptococci colonization in pregnant women

BackgroundGroup-B streptococci (GBS) colonization in pregnant women could be a source of serious neonatal infections, such as meningitis and sepsis; therefore, a rapid and accurate method for detection is needed for early prophylaxis. Conventional culture is a common method used for the detection of GBS. However, polymerase-chain reaction (PCR)-based assay may be another method for detection of GBS colonization.ObjectiveTo evaluate the performance of the PCR as a method for detection of GBS (Strept. agalactiae) colonization in the urogenital tract of pregnant women and to detect its prevalence among these women.MethodsThe present study was done at the Department of Clinical Pathology and Department of Obstetrics/Gynecology, Menoufia University Hospitals, during the period from June 2020 to June 2021. In this study, vaginal and perianal swabs were collected from 250 pregnant women, between the 35th and 37th weeks of pregnancy, who attended the Department of Obstetrics and Gynecology for perinatal care. Their ages ranged from 20 to 40 years. GBS was detected by culture VITEK2 and PCR was done.ResultsThe prevalence of colonization in pregnant women with GBS was 20%. The sensitivity and specificity of PCR and culture were 99% and 98%, respectively.ConclusionsPCR technique is a rapid, specific, and sensitive diagnostic tool for GBS detection. So, PCR could be used as a rapid test for the detection of GBS colonization in pregnant women. The sensitivity and specificity of automated culture were 99.8% and 89.2%, respectively, so it is also a rapid and specific method for the detection of GBS colonization.

Somatic mutation detection efficiency in EGFR: a comparison between high resolution melting analysis and Sanger sequencing

BackgroundHigh resolution melting curve analysis is a cost-effective rapid screening method for detection of somatic gene mutation. The performance characteristics of this technique has been explored previously, however, analytical parameters such as limit of detection of mutant allele fraction and total concentration of DNA, have not been addressed. The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in somatic mutations of the EGFR gene in non-small cell lung cancer.MethodsThe minor allele fraction of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM to determine the limit of detection. The mutant and wildtype allele fractions were validated by multiplex allele-specific real-time PCR. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing.ResultsThe limit of detection of minor allele fraction of exon 19 mutation was 1% with sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Multiplex allele-specific real-time PCR revealed that the wildtype DNA did not impede the amplification of mutant allele in mixed DNA assays. All mutation positive samples detected by Sanger sequencing, were also detected by HRM. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, were not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100 and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%.ConclusionThe comparative series study suggests that HRM is a modest initial screening test for somatic mutation detection of EGFR, which must further be confirmed by Sanger sequencing. With the modification of annealing temperature of initial PCR, the limit of detection of Sanger sequencing can be improved.

Combining genetically-encoded biosensors with high throughput strain screening to maximize erythritol production in Yarrowia lipolytica

Erythritol is an important sweetener ingredient and chemical precursor for synthesizing materials with phase transition behavior. Commercial erythritol is primarily produced by industrial fermentation. Further strain engineering necessitates the development of high throughput screening method for rapid detection and screening of mutant strain libraries. In this work, we took advantage of the erythritol-responsive transcription factor EryD, and constructed a sensor-regulator system for rapid screening and characterization of erythritol overproducers. We configured the optimal architecture of the EryD sensor-regulator construct with improved sensitivity, specificity and dynamic response range. Coupled with mutagenesis and strain screening based on biosensors, we rapidly screened and characterized a strain library containing 1152 mutants derived from combined UV and ARTP mutagenesis, in a relatively short period of time (1 week). The optimal strain produced more than 148 g/L erythritol in bench-top reactors. This work provides a reference for other metabolic engineering researchers to develop industrially-relevant strains. The reported framework enables us to rapidly improve strain performance and engineer efficient microbial cell factories for industrial applications.

Phenolic content as an indicator of tolerance of cowpea seedlings to Sclerotium rolfsii

The role of phenolics in plant tolerance to pathogen infection is well documented. The objective of the present preliminary investigation was to study phenolic metabolites involved in the tolerance or susceptibility of cowpea (Vigna unguiculata Walp.) cultivars to Sclerotium rolfsii Sacc. and to use their presence as a possible screening tool. Total, free acid, ester-bound and cell wall-bound phenolics of 10 cowpea cultivars were quantified. In healthy seedlings, the tolerant cultivars displayed the higher phenol content than the susceptible cultivars. In S. rolfsii infected seedlings, the highest increase was found from 48 h after inoculation. The net effect of inoculation was a 630% increase in total phenolics (soluble and insoluble) in the stem of tolerant cultivars while the total phenolic content increased only by 212% in the stems of susceptible cultivars. Although, no significant difference (P = 0.05) was detected among cultivars, in terms of free acid phenolics, the amount of ester-bound and cell wall-bound phenolics significantly increased, therefore demonstrating a similar trend to the one observed for the total phenolic content. These preliminary results showed that the presence of phenolics before and after S. rolfsii infection may be used as a rapid screening method for detection of tolerance to S. rolfsii damping-off and stem rot of cowpea.

Comparative ovarian microarray analysis of juvenile hormone-responsive genes in water flea Daphnia magna: potential targets for toxicity.

The freshwater zooplankton Daphnia magna has been extensively employed in chemical toxicity tests such as OECD Test Guidelines 202 and 211. Previously, it has been demonstrated that the treatment of juvenile hormones (JHs) or their analogues to female daphnids can induce male offspring production. Based on this finding, a rapid screening method for detection of chemicals with JH-activity was recently developed using adult D. magna. This screening system determines whether a chemical has JH-activity by investigating the male offspring inducibility. Although this is an efficient high-throughput short-term screening system, much remains to be discovered about JH-responsive pathways in the ovary, and whether different JH-activators act via the same mechanism. JH-responsive genes in the ovary including developing oocytes are still largely undescribed. Here, we conducted comparative microarray analyses using ovaries from Daphnia magna treated with fenoxycarb (Fx; artificial JH agonist) or methyl farnesoate (MF; a putative innate JH in daphnids) to elucidate responses to JH agonists in the ovary, including developing oocytes, at a JH-sensitive period for male sex determination. We demonstrate that induction of hemoglobin genes is a well-conserved response to JH even in the ovary, and a potential adverse effect of JH agonist is suppression of vitellogenin gene expression, that might cause reduction of offspring number. This is the first report demonstrating different transcriptomics profiles from MF and an artificial JH agonist in D. magna ovary, improving understanding the tissue-specific mode-of-action of JH. Copyright © 2016 John Wiley & Sons, Ltd.

DETECTION OF MATERNAL COLONIZATION OF GROUP B STREPTOCOCCUS BY PCR TARGETING Cfb AND ScpB GENES

Group B Streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Molecular based tests, such as polymerase chain reaction (PCR), can detect GBS within hours and can be used intrapartum allowing for selective intrapartum antibiotic prophylaxis (IAP) in women carrying GBS. The aim of this work was to evaluate PCR as a rapid screening method for detection of maternal colonization of GBS compared to culture. Vaginal/rectal swabs were collected from 120 pregnant women at 35-37 weeks of gestation and cultured on CNA medium. GBS was identified by gram staining and catalase, hippurate and CAMP tests and confirmed by latex agglutination for GBS antigens. PCR was done using two assays; one targeting the cfb gene and the other targeting the scpB gene. Results revealed thatGBS colonization was detected in 15%, 23.3% and 21.7% of pregnant women by culture, cfb PCR assay and scpB PCR assay respectively. cfb PCR assay showed 100% sensitivity and 90.2% specificity whereas scpB PCR assay showed 94.4% sensitivity and 91.2% specificity. PCR could detect GBS genome at a concentration of as low as 10-2 for cfb PCR and 10-3 for scpB PCR. In conclusion, PCR is a rapid, specific and sensitive tool for detection of maternal colonization of GBS. PCR assay targeting scpB gene is more sensitive than that targeting cfb gene.

High resolution melting analysis: a rapid and accurate method to detect CALR mutations.

BackgroundThe recent discovery of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without JAK2/MPL mutations has emerged as a relevant finding for the molecular diagnosis of these myeloproliferative neoplasms (MPN). We tested the feasibility of high-resolution melting (HRM) as a screening method for rapid detection of CALR mutations.Methods CALR was studied in wild-type JAK2/MPL patients including 34 ET, 21 persistent thrombocytosis suggestive of MPN and 98 suspected secondary thrombocytosis. CALR mutation analysis was performed through HRM and Sanger sequencing. We compared clinical features of CALR-mutated versus 45 JAK2/MPL-mutated subjects in ET.ResultsNineteen samples showed distinct HRM patterns from wild-type. Of them, 18 were mutations and one a polymorphism as confirmed by direct sequencing. CALR mutations were present in 44% of ET (15/34), 14% of persistent thrombocytosis suggestive of MPN (3/21) and none of the secondary thrombocytosis (0/98). Of the 18 mutants, 9 were 52 bp deletions, 8 were 5 bp insertions and other was a complex mutation with insertion/deletion. No mutations were found after sequencing analysis of 45 samples displaying wild-type HRM curves. HRM technique was reproducible, no false positive or negative were detected and the limit of detection was of 3%.ConclusionsThis study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations.

Rapid detection of rifampicin, isoniazid and streptomycin resistance in Mycobacterium tuberculosis clinical isolates by high-resolution melting curve analysis

This study was carried out to evaluate high-resolution melting (HRM) curve analysis assay for detection of mutations in three drug resistance-associated genes of Mycobacterium tuberculosis. Clinical isolates of Myco.tuberculosis phenotypically resistant to rifampicin (n=29), isoniazid (n=35) and streptomycin (n=34) were analysed for mutations in rpoB, katG and rpsL genes, respectively, by HRM curve analysis and DNA sequencing. HRM curve assay resulted in 11 clearly distinguishable melt curves denoting eight types of mutations responsible for drug resistance. For the three drugs, respectively, the sensitivity of HRM curve assay was found to be 93·1, 80 and 61·8% compared to the phenotypic resistance patterns, and 93·1, 93·3 and 100% in comparison with the DNA sequencing. The sensitivity and specificity of HRM curve assay was found to be comparable to DNA sequencing. The assay offers the advantage of high throughput, single step, rapid work flow and cost effectiveness and can be utilized as a rapid screening method for detection of drug-resistant tuberculosis. HRM curve assay may prove to be an important tool for the development of rapid molecular diagnostic assays for detection of mutation-based drug resistance.

Multicenter Evaluation of the LightCycler MRSA Advanced Test, the Xpert MRSA Assay, and MRSASelect Directly Plated Culture with Simulated Workflow Comparison for the Detection of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs

Rapid detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) followed by appropriate infection control procedures reduces MRSA infection and transmission. We compared the performance and workflow of two Food and Drug Administration-approved nucleic acid amplification assays, the LightCycler MRSA Advanced Test and the Xpert MRSA test, with those of directly plated culture (MRSASelect) using 1202 nasal swabs collected at three U.S. sites. The sensitivity of the LightCycler test (95.2%; 95% CI, 89.1% to 98.4%) and Xpert assay (99%; 95% CI, 94.8% to 100%) did not differ compared with that of culture; the specificity of the two assays was identical (95.5%; 95% CI, 94.1% to 96.7%) compared with culture. However, sequencing performed on 71 samples with discordant results among the three methods confirmed the presence of MRSA in 40% of samples that were positive by both molecular methods but negative by culture. Workflow analysis from all sites including batch runs revealed average hands-on sample preparation times of 1.40, 2.35, and 1.44 minutes per sample for the LightCycler, Xpert, and MRSASelect methods, respectively. Discrete event simulation analysis of workflow efficiencies revealed that the LightCycler test used less hands-on time for the assay when greater than eight batched samples were run. The high sensitivity and specificity, low hands-on time, and efficiency gains using batching capabilities make the LightCycler test suitable for rapid batch screening of MRSA colonization.

Disposable sensors for rapid screening of mutated genes

A screening method for rapid detection of gene mutations directly in polymerase chain reaction (PCR) of genomic DNA is described. The method involves the development of a disposable screen-printed gold electrode modified with a thiolated capture probe directly obtained from denaturated PCR genomic DNA, which recognizes (by hybridization) its fully complementary sequence (wild type), giving a signal, whereas no signal is obtained for single-mismatched target (mutant). The detection of the hybridization event is achieved by changes in the metal redox center electroactivity of the complex [Ru(NH(3))(5) L](2+), where L is [3-(2-phenanthren-9-yl-vinyl)-pyridine], at -0.200 V. This complex binds to double-stranded DNA in a very selective form. The method allows discrimination between the wild type and the mutant of gene MRP3 directly in large PCR amplicons extracted from blood cells, without the need to use either synthetic probes or labeled targets. The mutation involves the presence of a single-nucleotide polymorphism (SNP) at base 54 of a 145-base-pair sequence from exon 21 of gene MRP3. Since the presence of this SNP might lead to a variety of hereditary liver disorders, its identification in a rapid and easy form may provide novel therapeutic targets for the future. The screening method proposed has excellent signal reproducibility, with a relative standard deviation of 10%. In addition, with the method developed as little as 6.6 ng/muL PCR product can be detected.

Strip-based immunoassay for rapid detection of thiabendazole

There is increased interest in the investigation and implementation of rapid screening methods for detection of pesticide residues. This study reports development of an immunostrip test for thiabendazole detection based on indirect competitive principle using carbon particles as a label. Nitrocellulose membrane strip was coated with a thiabendazole-protein conjugate in the defined test zone. In flow of an antibody-carbon complex and thiabendazole along the strip, the intensity of black colour formed in the test line reflected the thiabendazole concentration and semi-quantitative estimation could be carried out visually. The optimized test was accomplished within 10 min and the visual detection limit was achieved 0.25 ng mL −1 of standard sample. Moreover, immunostrip was evaluated quantitatively using scanning densitometry. Based on standard curve, the detection limit of the proposed test was as low as 0.08 ± 0.03 ng mL −1 with an IC 50 value of 0.60 ± 0.08 ng mL −1 and a linear working range of 0.11–4.13 ng mL −1. Results of testing precision, stability, and specificity demonstrated that the assay provided a reliable performance. This immunostrip was applied to analysis of spiked fruit juices in range of 0.05–5 mg L −1. Matrix interferences were avoided by simple dilution of samples. Both visual and instrumental evaluations indicated a good agreement with results obtained by ELISA. Recoveries from juices were from 81.9 to 123.6% and relative standard deviations ranged from 9.9 to 19.3%. The developed strip offers potential as a useful rapid and simple method for screening of thiabendazole in fruit juices at levels far below the maximum residue limits.

A PCR-based screening method for rapid detection and genetic counseling in Fragile-X syndrome

A PCR-based screening method for rapid detection and genetic counseling in Fragile-X syndrome

Mapping cyanobacterial blooms in the Great Lakes using MODIS

Toxin-producing Cyanobacteria have been documented in Lake Erie and Ontario in the last several years. We developed algorithms to discriminate potentially toxic cyanobacterial blooms from other harmless phytoplankton blooms and to extract relative phycocyanin abundances from Moderate Resolution Imaging Spectrometer (MODIS) satellite data. Lee's quasi-analytical algorithm was used to calculate total absorption and backscatter from the 250 m, 500 m and 1 km bands of MODIS scenes. A non-negative least square algorithm was then utilized to discern relative concentrations of Chlorophyta (green algae), phycocyanin-rich Cyanobacteria (blue-green algae), and colored dissolved organic matter and suspended sediments combined in lake waters using published absorption spectra for these components. MODIS-derived cyanobacterial concentrations and/or bloom distributions from 10 scenes acquired in the summers of 2004 and 2005 were successfully verified against contemporaneous calibrated measurements of pigments that were acquired from measurements made using continuous fluorimetric measurements of surface water (1 m depth) from six cruises, and three additional cyanobacterial blooms reported in the scientific literature between 2002 and 2006. These results demonstrate that this methodology could be used to develop a cost-effective practical screening method for rapid detection and warning of potentially toxic cyanobacterial blooms in the lower Great Lakes.

Octadecylsilane-modified silicas in the adsorption of toluene

A series of octadecylsilane-modified silicas were prepared by sol-gel and grafting methods. Carbon contents and octadecyl chain conformations were shown to depend on the preparative route. Grafting engenders a low carbon content and a liquid-like chain conformation, while the sol-gel method affords a much higher carbon content and a crystalline conformation. The relationships between the toluene adsorption of the hybrid silicas and their chain conformations, their carbon contents and their textural characteristics are discussed. These sorbents, when used in combination with ultraviolet diffuse reflectance spectroscopy (UV DRS), can be employed as a rapid screening method for detection of aromatic compounds in water and air environmental matrices.

A Polymerase Chain Reaction Screening Method for Rapid Detection of Microsatellites in Bacterial Artificial Chromosomes

Standard protocols aimed at identifying subclones of interest from bacterial artificial chromosomes (BACs) include the use of hybridization methods that are time consuming and often require the use of radioactive isotopes. Through our efforts to identify microsatellites in BACs from rainbow trout (Oncorhynchus mykiss) we have developed a nonradioactive polymerase chain reaction (PCR)-based screening technique to select microsatellites containing subclones for marker development. Two BACs were subcloned and screened by PCR using a vector-specific primer and a mix of microsatellite repeat primers. The subclones were then sequenced to evaluate the efficiency of the PCR screening method. Correlation between positive PCR amplification and presence of microsatellites varied between the two BACs (21.9% and 71.4%), but still a sufficient number of subclones were identified to enable design and optimization of microsatellite markers.

Evaluation of a rapid screening method for detection of antimicrobial resistance in the commensal microbiota of the gut

The assessment of antimicrobial resistance among commensal bacteria is an indicator of the spread of antimicrobial resistance. Rapid screening methods for detection of antimicrobial-resistant faecal Escherichia coli directly on MacConkey plates have been successfully adopted but suffer from lack of standardisation. The purpose of this study was to evaluate a direct plating method (DPM) for detection of antimicrobial-resistant faecal E. coli and to compare it with a conventional method. Faecal samples were collected from 71 healthy children from Peru and Bolivia. In the DPM, a faecal swab was directly plated onto a MacConkey agar plate and antimicrobial disks were applied onto the seeded plate. Raw data were obtained by direct reading of the plate and were subjected to confirmatory analysis. Good concordance between the DPM and a conventional method was observed in detecting carriage of resistant E. coli, with a higher sensitivity for the DPM. Analysis of the results allowed interpretive criteria to be defined for DPM raw data. The DPM showed good sensitivity and specificity at very low cost (ten times cheaper than the conventional method) to investigate the faecal carriage of drug-resistant E. coli. It may represent a useful tool to conduct large-scale resistance surveillance studies and to monitor resistance control programmes cost effectively, particularly in low-resource countries.

Protein microarray using α-amino acids as metal tags on chips

Procedures for synthesizing α-amino acids on a chip for coordination with transitional metal ions and a His-Tagged protein have been successfully developed as a stable protein microarray. Using the recombinant His-Tagged 3CL-protease (3CL pro) as a model for attachment to chips containing d-/ l-Glu, Asp, Orn, Ser via different transitional metal ions, it was found that the Orn chip was the best of affinity binding and stability by which Zn 2+ was the best metal ion for affinity while Co 2+ was the best metal ion for stability. Thus, this protein microarray can be alternatively used as a high throughput screening method for rapid detection against SARS CoV 3CL pro and/or efficient purification of other Tagged proteins.

Rapid screening method for detection of bacteria in platelet concentrates.

Public awareness has long focused on the risks of the transmission of viral agents through blood product transfusion. This risk, however, pales in comparison to the less publicized danger associated with the transfusion of blood products contaminated with bacteria, in particular, platelet concentrates. Up to 1,000 cases of clinical sepsis after the transfusion of platelet concentrates are reported annually in the United States. The condition is characterized by acute reaction symptoms and the rapid onset of septicemia and carries a 20 to 40% mortality rate. The urgent need for a method for the routine screening of platelet concentrates to improve patient safety has long been recognized. We describe the development of a rapid and highly sensitive method for screening for bacteria in platelet concentrates for transfusion. No culture period is required; and the entire procedure, from the time of sampling to the time that the final result is obtained, takes less than 90 min. The method involves three basic stages: the selective removal of platelets by filtration following activation with a monoclonal antibody, DNA-specific fluorescent labeling of bacteria, and concentration of the bacteria on a membrane surface for enumeration by solid-phase cytometry. The method offers a universal means of detection of live, nondividing, or dead gram-negative and gram-positive bacteria in complex cellular blood products. The sensitivity is higher than those of the culture-based methods available at present, with a detection limit of 10 to 10(2) CFU/ml, depending upon the bacterial strain.

DNA Comet Assay––a rapid screening method for detection of irradiated cereals and tree nuts

Abstract Whole grains of some important cereals (wheat, buckwheat, maize, millet and oat) and some whole tree nuts (almonds, Brazil nuts, cashew nuts, peanuts, walnuts, hazelnuts, pistachio nuts and pine nuts) have been investigated for the identification of radiation treatment using microgel electrophoresis of single cells. This DNA Comet Assay is proposed as a rapid screening method for detection of irradiated foods, but is not radiation specific. Cereals and nuts were exposed to radiation doses of 0.5, 1 and 3 or 5 kGy covering the range for insect/pest disinfestation or for microbial control. After electrophoresis, irradiated cells from buckwheat, maize, millet and oat, and also from almonds, peanuts, walnuts and hazelnuts showed comets indicating stretching of fragmented DNA towards the anode. The size and shape of the comets were dose dependent. Non-irradiated samples of all cereals showed intact cells/nuclei in form of round stains or with short faint tails except for wheat, which showed only comets also in non-irradiated samples. For nuts like Brazil, cashew and pistachio nuts, suitable DNA material could not be extracted. In case of pine nuts, very few round intact cells were observed along with a majority of comets, making the screening difficult. Thus comet assay can be successfully used as a screening method for detection of radiation treatment of several cereals and nuts, however, the assay was not satisfactory for wheat and for Brazil nuts, cashew nuts, and pistachio nuts.