scholarly journals PCR-Dipstick Chromatography for Differential Detection of Carbapenemase Genes Directly in Stool Specimens

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Rathina Kumar Shanmugakani ◽  
Yukihiro Akeda ◽  
Norihisa Yamamoto ◽  
Noriko Sakamoto ◽  
Hideharu Hagiya ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Hybridization ◽  
Genomic Dna ◽  
Detection System ◽  
High Sensitivity ◽  
Visual Observation ◽  
Differential Detection ◽  
Pcr Products ◽  
Stool Specimens ◽  
Differential Identification

ABSTRACT A PCR-dipstick chromatography technique was designed and evaluated for differential identification of bla NDM, bla KPC, bla IMP, and bla OXA-48 carbapenemase genes directly in stool specimens within 2 h. It is a DNA-DNA hybridization-based detection system where PCR products can be easily interpreted by visual observation without electrophoresis. The PCR-dipstick showed high sensitivity (93.3%) and specificity (99.1%) in directly detecting carbapenemase genes in stool specimens compared with multiplex PCR for genomic DNA of the isolates from those stool specimens.

Electrochemical detection of pathogenic bacteria by using a glucose dehydrogenase fused zinc finger protein

2014 ◽  
Vol 6 (14) ◽  
pp. 4991-4994 ◽  
Author(s):  
Jinhee Lee ◽  
Atsuro Tatsumi ◽  
Koichi Abe ◽  
Wataru Yoshida ◽  
Koji Sode ◽  
...  
Keyword(s):  
Electrochemical Detection ◽  
Zinc Finger ◽  
Genomic Dna ◽  
Pathogenic Bacteria ◽  
Zinc Finger Protein ◽  
Detection System ◽  
Glucose Dehydrogenase ◽  
Finger Protein ◽  
Pcr Products ◽  
Probe Hybridization

We developed an electrochemical detection system for pathogenic bacteria by utilizing a glucose dehydrogenase-fused zinc finger protein (ZF-GDH), which could detect PCR products electrochemically without the need for DNA probe hybridization. Using ZF-GDH, we could specifically detect 10 copies of genomic DNA derived fromEscherichia coliO157.

scholarly journals Thermodynamics and kinetics guided probe design for uniformly sensitive and specific DNA hybridization without optimization

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Xin Chen ◽  
Na Liu ◽  
Liquan Liu ◽  
Wei Chen ◽  
Na Chen ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Dna Hybridization ◽  
Genomic Dna ◽  
Inverse Correlation ◽  
High Sensitivity ◽  
Strand Exchange ◽  
Probe Design ◽  
Dna Testing ◽  
Thermodynamics And Kinetics ◽  
Human Genomic

Abstract Sensitive and specific DNA hybridization is essential for nucleic acid chemistry. Competitive composition of probe and blocker has been the most adopted probe design for its relatively high sensitivity and specificity. However, the sensitivity and specificity were inversely correlated over the length and concentration of the blocker strand, making the optimization process cumbersome. Herein, we construct a theoretical model for competitive DNA hybridization, which disclose that both the thermodynamics and kinetics contribute to the inverse correlation. Guided by this, we invent the 4-way Strand Exchange LEd Competitive DNA Testing (SELECT) system, which breaks up the inverse correlation. Using SELECT, we identified 16 hot-pot mutations in human genome under uniform conditions, without optimization at all. The specificities were all above 140. As a demonstration of the clinical practicability, we develop probe systems that detect mutations in human genomic DNA extracted from ovarian cancer patients with a detection limit of 0.1%.

A Multiplex PCR Assay Mediated by Universal Primers for the Detection of Adulterated Meat in Mutton

2019 ◽  
Vol 82 (2) ◽  
pp. 325-330 ◽  
Author(s):  
WANWAN LIU ◽  
XIAONAN WANG ◽  
JING TAO ◽  
BANGSHENG XI ◽  
MAN XUE ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Detection System ◽  
Meat Products ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Food Samples ◽  
Detection Sensitivity ◽  
Universal Primers ◽  
Multiplex Pcr Assay ◽  
Multiple Pcr

ABSTRACT This study aimed to establish a multiplex PCR detection system mediated by “universal primers,” which would be able to determine whether mutton meat contained nonmutton ingredients from rats, foxes, and ducks. Based on the sequence variation of specific mitochondrial genes, nine different multiplex PCR primers were designed, and four kinds of meat products were rapidly identified by electrophoresis using an optimized multiplex PCR system based on the molecular weight differences of the amplified products. Multiplex PCR applications optimized for meat food source from food samples for testing was used to verify the accuracy of the identification method. The results showed that the primers in multiple PCR system mediated by universal primers could be used for the rapid identification of rat, fox, duck, and sheep meat in mutton products, and the detection sensitivity could reach 0.05 ng/μL. The identification of food samples validated the practical value of this method. Therefore, a multiplex PCR system mediated by universal primers was established, which can be used to quickly identify the origin of animal ingredients from rats, foxes, and ducks in mutton products.

Abstract 4336: Interrogating the Human Myocardial Interstitium during Myocardial Arrest and Reperfusion: Dynamic Changes in Cytokines and Protease Activity

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Robert E Stroud ◽  
Christine N Koval ◽  
Isabelle Gengler ◽  
Anne M Deschamps ◽  
John S Ikonomidis ◽  
...  
Keyword(s):  
Detection System ◽  
Ischemia Reperfusion ◽  
Dynamic Assessment ◽  
High Sensitivity ◽  
Fluorescent Detection ◽  
Suspension Array ◽  
Cytokine Analysis ◽  
And Function ◽  
Inflammatory Molecules ◽  
Pulmonary Bypass

Background. Cytokines, such as the interleukins (IL1β, IL2, IL6) and tumor necrosis factor (TNF) can modulate myocardial structure and function with ischemia/reperfusion (I/R) but dynamic assessment of these biological molecules within the human myocardial interstitium with I/R has not been performed, and the inter-relationship to matrix metalloproteinases activity (MMPact) remains unexplored. Accordingly, a fluorogenic microdialysis method was used to simultaneously measure myocardial interstitial cytokine levels and MMPact in patients during and following I/R. Methods . MMPact was measured in patients (n=13) undergoing cardio-pulmonary bypass (CPB) at baseline, during myocardial arrest and CPB (on-CPB), and immediately following reperfusion and separation from CPB (post-CPB) by a validated in-line microdialysis fluorescent detection system. Myocardial interstitial fluid was subjected to cytokine analysis by high sensitivity multiplex suspension array. Results . Interstitial MMPact increased by over 30% post-CPB and was accompanied by a specific change in cytokine profiles (Figure ). The classical pro-inflammatory molecules such as TNF and IL6 were either not detectable or unchanged, whereas IL1β and IL2 which can be proinflammatory, were increased. Conclusions. These unique results demonstrated that a dynamic cytokine signature occurs within the human myocardial interstitium following I/R and is temporally related to heightened MMP activity. Direct interrogation of the human myocardial interstitium may provide a unique insight into critical signaling pathways which may evoke adverse structural and functional events following I/R.

Compact Detection System for High Sensitivity Hydrogen Profiling of Materials by Nuclear Reaction Analysis

2009 ◽  
Author(s):  
Daniel Keith Marble ◽  
Ben Urban ◽  
Jose Pacheco ◽  
Floyd D. McDaniel ◽  
Barney L. Doyle
Keyword(s):  
Nuclear Reaction ◽  
Detection System ◽  
High Sensitivity ◽  
Nuclear Reaction Analysis

Genomic variability within laboratory and wild subspecies of Heligmosomoides polygyrus

1994 ◽  
Vol 68 (2) ◽  
pp. 93-96 ◽  
Author(s):  
M.A. Abu-Madi ◽  
A.P. Reid ◽  
J.W. Lewis ◽  
W.M. Hominick
Keyword(s):  
Restriction Endonuclease ◽  
Ribosomal Dna ◽  
Dna Hybridization ◽  
Genomic Dna ◽  
Dna Probes ◽  
Heligmosomoides Polygyrus ◽  
Banding Patterns ◽  
Genomic Variability

AbstractGenomic DNA extracted from laboratory and wild subspecies of the trichostrongyle nematode Heligmosomoides polygyrus were compared using RFLP and DNA/DNA hybridization techniques. Eight restriction endonuclease digests of the genomic DNA of the two subspecies were hybridized with heterologous ribosomal DNA probes and the total radio-isotope labelled DNA of the laboratory subspecies. DNA hybridization of the two subspecies of H. polygyrus yielded different banding patterns when probed with the rDNA clones in Pvu II digests and when total genomic DNA was used as the probe in Hind III and Pvu II digests. The remaining hybridization profiles of both subspecies were identical.

scholarly journals A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

2003 ◽  
Vol 17 (2) ◽  
pp. 142-146 ◽  
Author(s):  
José Freitas Siqueira Júnior ◽  
Isabela das Neves Rôças
Keyword(s):  
16S Rdna ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Nested Polymerase Chain Reaction ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root ◽  
Pcr Products ◽  
Species Specific

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

scholarly journals Multiplex PCR for direct identification of Campylobacter spp. in human and chicken stools

2007 ◽  
Vol 56 (10) ◽  
pp. 1350-1355 ◽  
Author(s):  
Aisha Al Amri ◽  
Abiola C. Senok ◽  
Abdulrahman Yusuf Ismaeel ◽  
Ali E. Al-Mahmeed ◽  
Giuseppe A. Botta
Keyword(s):  
Multiplex Pcr ◽  
Direct Detection ◽  
False Negative ◽  
Enteric Bacteria ◽  
Enzymic Activity ◽  
Campylobacter Coli ◽  
Biochemical Tests ◽  
Direct Evaluation ◽  
Stool Specimens ◽  
Culture Negative

Differentiation between Campylobacter jejuni and Campylobacter coli is problematic in clinical specimens due to fastidious growth requirements and limited biochemical tests. This study describes a rapid, multiplex PCR protocol for the direct detection and differentiation of C. jejuni and C. coli in stools. An evaluation was carried out of this multiplex protocol based on the detection of cadF (genus specific), and hipO (C. jejuni) and asp (C. coli) genes, using stool from patients with Campylobacter enteritis and chicken. Protocol sensitivity was assessed and specificity determined using a panel of enteric bacteria, and evaluation of 30 diarrhoeic stool specimens culture negative for Campylobacter. Of the 114 specimens (54 human and 60 chicken) evaluated by the protocol, 70 (61.4 %) were identified as C. jejuni, 35 (30.7 %) as C. coli and 9 (7.9 %) as a mixed infection/colonization with both species. All mixed infections were identified as C. jejuni by culture. Among the stool specimens that were culture negative for Campylobacter, two (6.7 %) were C. jejuni positive by multiplex PCR. The protocol sensitivity limit was 0.015–0.016 ng C. jejuni and C. coli DNA μl−1 in the specimen. There was no cross-reaction with the reference strains assessed. Comparison of hippurate test and multiplex PCR demonstrated 17 isolates with false-positive hippurate enzymic activity and 7 with false-negative activity. This rapid protocol (turnaround time 6 h) is highly sensitive and specific for direct evaluation of stool for these pathogens. It has significant application for routine clinical diagnostic and epidemiological purposes.

scholarly journals Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vβ-Chain Repertoire

2005 ◽  
Vol 12 (4) ◽  
pp. 477-483 ◽  
Author(s):  
Sanjit Fernandes ◽  
Surendra Chavan ◽  
Vivek Chitnis ◽  
Nina Kohn ◽  
Savita Pahwa
Keyword(s):  
T Cell ◽  
Multiplex Pcr ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Clinical Samples ◽  
Cdr3 Length ◽  
Pcr Products ◽  
Pcr Method ◽  
T Cell Receptor Vβ

ABSTRACTRationale: evaluation of the T-cell receptor (TCR) Vβ-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR Vβ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream Vβ primers instead of the conventionally labeled downstream Cβ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a Cβ primer and an Omniscript RT kit. The 24 Vβ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 Vβ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled Cβ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: Vβ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled Vβ primers. The resolution was superior to that obtained with the labeled Cβ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the Cβ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor Vβ-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR Vβ repertoire analyses in clinical trials.

scholarly journals Prototype Sistem Deteksi Partial Discharge Pada Isolasi Kabel Menggunakan Sensor Microphone (Prototype Of Partial Discharge Detection System In Cable Isolation Using Microphone Sensor)

2019 ◽  
Vol 3 (2) ◽  
pp. 206
Author(s):  
Mochamad Zaeynuri Setiawan ◽  
Fachrudin Hunaini ◽  
Mohamad Mukhsim
Keyword(s):  
Detection System ◽  
Partial Discharge ◽  
Measurement Data ◽  
Negative Ions ◽  
High Sensitivity ◽  
Sound Waves ◽  
Cable Insulation ◽  
Insulation Failure ◽  
Measurement Results ◽  
Discharge Detection

The phenomenon that often arises in a substation is the problem of partial discharge in outgoing cable insulation. Partial discharge is a jump of positive and negative ions that are not supposed to meet so that it can cause a spark jump. If a partial discharge is left too long it can cause insulation failure, the sound of snakes like hissing and the most can cause a flashover on the outgoing cable. Then a partial discharge detection prototype was made in the cable insulation in order to anticipate the isolation interference in the outgoing cable. Can simplify the work of substation operators to check the reliability of insulation on the outgoing side of each cubicle. So it was compiled as a method for measuring sound waves caused by partial discharge in the process of measuring using a microphone sensor, the Arduino Mega 2560 module as a microcontroller, the LCD TFT as a monitoring and the MicroSD card module as its storage. The microphone sensor is a sensor that has a high sensitivity to sound, has 2 analog and digital readings, and is easily designed with a microcontroller. Basically the unit of measure measured at partial discharge is Decibels. The results of the prototype can be applied to the cubicle and the way it works is to match the prototype to the outgoing cubicle cable then measure from the cable boots connector to the bottom of the outgoing cable with a distance of 1 meter. Then the measurement results will be monitored on the TFT LCD screen in the form of measurement results, graphs and categories on partial discharge. In this design the measurement data made by the microphone can be stored with microSD so that it can make an evaluation of partial discharge handling in outgoing cable insulation.

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