scholarly journals Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovorain Asymptomatic Plant Material

2000 ◽  
Vol 66 (5) ◽  
pp. 2071-2078 ◽  
Author(s):  
Pablo Llop ◽  
Anna Bonaterra ◽  
Javier Peñalver ◽  
María M. López
Keyword(s):  
Nested Pcr ◽  
Plant Material ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Sample Volume ◽  
Small Sample ◽  
Asymptomatic Plant ◽  
Specificity And Sensitivity ◽  
Closed Tube ◽  
Pure Cultures

ABSTRACT A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.

scholarly journals Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255914
Author(s):  
Monia Ardhaoui ◽  
Emna Ennaifer ◽  
Anna Christina De Matos Salim ◽  
Flávio Marcom Gomez ◽  
Thalja Laasili ◽  
...  
Keyword(s):  
Nested Pcr ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Multiple Infection ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Hpv Genotyping ◽  
Genotyping Method ◽  
Specificity And Sensitivity ◽  
Hpv Types

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

scholarly journals Specificity and sensitivity of three PCR-based methods for detection of Erwinia amylovora in pure culture and plant material

Genetika ◽  
2019 ◽  
Vol 51 (3) ◽  
pp. 1039-1052
Author(s):  
Milan Ivanovic ◽  
Nemanja Kuzmanovic ◽  
Katarina Gasic ◽  
Andjelka Prokic ◽  
Nevena Zlatkovic ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Pure Culture ◽  
Plant Material ◽  
Erwinia Amylovora ◽  
Inhibitory Effect ◽  
Bacterial Cells ◽  
Conventional Pcr ◽  
Specificity And Sensitivity ◽  
Pure Cultures ◽  
Sensitivity Specificity

Three PCR methods, referred in this study as ?conventional?, ?nested? and ?chromosomal? PCR and suggested for routine detection of Erwinia amylovora in pure culture and plant material, were evaluated according to their specificity and sensitivity. Specificity of PCR methods was analyzed by using 42 strains of E. amylovora, originating from different locations and plant species, with diverse PFGE profiles, representing distant populations of the pathogen. Sensitivity of PCR protocols in pure culture was studied by using nine different concentrations of E. amylovora in sterile ultrapure water as a template in PCR reactions. In order to study inhibitory effect of plant DNA and other inhibitors on sensitivity of the three PCR methods bacterial dilutions were mixed with plant macerate of pear, apple and quince prior to the PCR reaction. In specificity assays, tested PCR protocols were able to detect all E. amylovora strains regardless of the host of the strain, its origin or PFGE group, indicating primer specificity. On the other hand, sensitivity among tested methods varied, depending on bacterial concentration and selected plant material used in the PCR. When working with pure cultures nested PCR showed the greatest sensitivity by detecting 1.9 bacterial cells per PCR reaction, followed by detection limit of 9.5 cells per PCR reaction with conventional PCR and 1.9?105 cells/PCR reaction with chromosomal PCR. In spiked samples plant inhibitors either did not affect or they decreased the sensitivity of the PCR reaction, depending on the protocol and/or type of plant macerate. In our experiments, inhibitors from pear and quince macerates did not affect sensitivity of nested PCR, while apple macerate reduced its sensitivity by a factor of 10. Conventional PCR protocol was able to detect 95 cells/PCR reaction in pear and apple macerate, but only 9.5?103 cells/PCR in quince macerate. Greatest decrease in sensitivity of the PCR method was observed in spiked samples with chromosomal PCR since bacterial DNA was not detected in each of the spiked samples. Our research shows that all three PCR protocols are specific for detection of E. amylovora, but nested PCR proved to be most sensitive when working with pure cultures and plant material.

scholarly journals X-Ray Fluorescence Analysis Applied to Small Samples

1977 ◽  
Vol 21 ◽  
pp. 171-185 ◽  
Author(s):  
J.M. Jaklevic ◽  
W.R. French ◽  
T.W. Clarkson ◽  
M.R. Greenwood
Keyword(s):  
Small Diameter ◽  
Fluorescence Analysis ◽  
High Sensitivity ◽  
Sample Volume ◽  
Small Sample ◽  
Small Samples ◽  
X Ray ◽  
Hair Samples ◽  
Fine Focus ◽  
Analysis System

We describe an adaptation of photon excited x-ray fluorescence analysis which is optimized for the analysis of small samples. A fine focus x-ray tube is used in conjunction with small diameter detector collimators in order to focus on a small sample volume with as high sensitivity as possible. Sample areas of less than 1 mm diameter can be analyzed with ppm detectability. In applications involving the analysis of human hair samples, a minimum detectable limit of 10 ppm Hg can be realized in a 1 mm long segment of a single hair in a counting time of 200 seconds. Simultaneous measurements of the sample mass can be obtained from the intensity of the incoherent scattering. An automated x-ray fluorescence analysis system using the technique for the scanning of elemental profiles in such hair samples will be described.

scholarly journals Sensitive and Specific Detection of Platelet-Derived and Tissue Factor–Positive Extracellular Vesicles in Plasma Using Solid-Phase Proximity Ligation Assay

TH Open ◽  
2018 ◽  
Vol 02 (03) ◽  
pp. e250-e260 ◽  
Author(s):  
Åsa Thulin ◽  
Junhong Yan ◽  
Mikael Åberg ◽  
Christina Christersson ◽  
Masood Kamali-Moghaddam ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Extracellular Vesicles ◽  
Solid Phase ◽  
Flow Cytometric Analysis ◽  
High Sensitivity ◽  
Small Sample ◽  
Proximity Ligation Assay ◽  
Clinical Samples ◽  
Proximity Ligation ◽  
Specificity And Sensitivity

AbstractExtracellular vesicles (EVs) derived from blood cells are promising biomarkers for various diseases. However, they are difficult to measure accurately in plasma due to their small size. Here, we demonstrate that platelet-derived EVs in plasma can be measured using solid-phase proximity ligation assay with high sensitivity and specificity using very small sample volume of biological materials. The results correlate well with high-sensitivity flow cytometry with the difference that the smallest EVs are detected. Briefly, the EVs are first captured on a solid phase, using lactadherin binding, and detection requires recognition with two antibodies followed by qPCR. The assay, using cholera toxin subunit-B or lactadherin as capture agents, also allowed detection of the more rare population of tissue factor (TF)-positive EVs at a concentration similar to sensitive TF activity assays. Thus, this assay can detect different types of EVs with high specificity and sensitivity, and has the potential to be an attractive alternative to flow cytometric analysis of preclinical and clinical samples. Improved techniques for measuring EVs in plasma will hopefully contribute to the understanding of their role in several diseases.

scholarly journals Dual-comb Photothermal Spectroscopy

2021 ◽  
Author(s):  
Qiang Wang ◽  
Zhen Wang ◽  
Hui Zhang ◽  
Shoulin Jiang ◽  
Yingying Wang ◽  
...  
Keyword(s):  
Gas Sensing ◽  
Spectroscopic Method ◽  
High Sensitivity ◽  
Fast Response ◽  
Sample Volume ◽  
Small Sample ◽  
Probe Laser ◽  
Spectral Measurements ◽  
Photothermal Spectroscopy ◽  
Fine Resolution

Abstract Dual-comb spectroscopy (DCS) has revolutionized optical spectroscopy by providing broadband spectral measurements with unprecedent resolution and fast response. Photothermal spectroscopy (PTS) offers an ultrasensitive and background-free gas sensing method, which is normally performed using a single-wavelength pump laser. The merging of PTS with DCS may enable a new spectroscopic method by taking advantage of both technologies, which has never been studied yet. Here, we report dual-comb photothermal spectroscopy (DC-PTS) by passing dual combs and a probe laser through a gas-filled anti-resonant hollow-core fiber, where the generated multi-heterodyne modulation of the refractive index is sensitively detected by an in-line interferometer. As an example, we have measured photothermal spectra of acetylene over 1 THz, showing a good agreement with the spectral database. Our proposed DC-PTS provides new opportunities for broadband gas sensing with super-fine resolution and high sensitivity, as well as with a small sample volume and compact configuration.

scholarly journals Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

2011 ◽  
Vol 31 (7) ◽  
pp. 575-578 ◽  
Author(s):  
Danielle C. Leal ◽  
Cláudio R. Madruga ◽  
Paulo F. de Matos ◽  
Bárbara M. P. da S. Souza ◽  
Carlos R. Franke
Keyword(s):  
Multiplex Pcr ◽  
Nested Pcr ◽  
Serological Test ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Conventional Pcr ◽  
Theileria Equi ◽  
Babesia Caballi ◽  
High Agreement ◽  
Laboratory Contamination

Conventional PCR (PCRTeq) for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc) for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq) for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128), but did not differ significantly from the M/PCRTeq-Bc (1:64). In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780) and moderate agreement with N/PCR-Teq (k = 0.562) and M/PCRTeq-Bc (k = 0.488). PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05), and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05). PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

scholarly journals Development of an on-chip detection of Zika virus and antibodies simultaneously using array of nanowells

2018 ◽  
Author(s):  
Touyana Semenova ◽  
Alexandria Voigt ◽  
William Donelan ◽  
Alek Aranyos ◽  
Janet Yamamoto ◽  
...  
Keyword(s):  
Zika Virus ◽  
High Sensitivity ◽  
High Specificity ◽  
Small Sample ◽  
Epifluorescence Microscopy ◽  
Clinical Samples ◽  
Serological Tests ◽  
Specificity And Sensitivity ◽  
Control And Prevention ◽  
On Chip

ABSTRACTZika virus (ZIKV) infections are an emerging health pandemic of significant medical importance. ZIKV appeared recently in the Americas from Africa via the South Pacific. The current outbreak has garnered attention by exhibiting unique characteristics of devastating neurodevelopmental defects in newborns of infected pregnant women. Current guidelines for ZIKV diagnostics developed by the Center of Diseases Control and Prevention (CDC) consist of nucleic acid testing, plaque reduction neutralization test (PRNT), and a serologic test for IgM detection. To better accommodate and comply with these guidelines, we developed a simultaneous on-chip detection of ZIKV and anti-ZIKV antibodies using an array of nanowells. Using on-chip microengraving, we were able to detect anti-ZIKV antibodies and their immunoglobulin isotypes. In parallel, applying on-chip real-time PCR with epifluorescence microscopy, we were able to quantify ZIKV viral load as low as one copy. To test clinical samples of patients at the postconvalescent stage, we analyzed samples from 8 patients. The on-chip nanowells could effectively identify antibodies that reacted against ZIKV envelope protein and their isotypes with high sensitivity and specificity. The small sample requirement with high specificity and sensitivity and combined molecular and serological tests could potentially be very advantageous and beneficial in accurate detection of Zika infection for better disease monitoring and management.

scholarly journals Universal Stokes’s nanomechanical viscometer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Komal Chaudhary ◽  
Pooja Munjal ◽  
Kamal P. Singh
Keyword(s):  
Electric Field ◽  
Real Time ◽  
Sample Volume ◽  
Capillary Waves ◽  
Small Sample ◽  
Medical Applications ◽  
Rapid Measurement ◽  
Single Lens ◽  
Universal Applicability ◽  
Small Sample Volume

AbstractAlthough, many conventional approaches have been used to measure viscosity of fluids, most methods do not allow non-contact, rapid measurements on small sample volume and have universal applicability to all fluids. Here, we demonstrate a simple yet universal viscometer, as proposed by Stokes more than a century ago, exploiting damping of capillary waves generated electrically and probed optically with sub-nanoscale precision. Using a low electric field local actuation of fluids we generate quasi-monochromatic propagating capillary waves and employ a pair of single-lens based compact interferometers to measure attenuation of capillary waves in real-time. Our setup allows rapid measurement of viscosity of a wide variety of polar, non-polar, transparent, opaque, thin or thick fluids having viscosity values varying over four orders of magnitude from $$10^{0}{-}10^{4}~\text{mPa} \, \text{s}$$ 10 0 - 10 4 mPa s . Furthermore, we discuss two additional damping mechanisms for nanomechanical capillary waves caused by bottom friction and top nano-layer appearing in micro-litre droplets. Such self-stabilized droplets when coupled with precision interferometers form interesting microscopic platform for picomechanical optofluidics for fundamental, industrial and medical applications.

scholarly journals Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein

Biosensors ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 4
Author(s):  
Donggee Rho ◽  
Seunghyun Kim
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Optical Cavity ◽  
Sample Volume ◽  
Small Sample ◽  
Label Free ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Cavity Structure

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

scholarly journals The associations between whole grain and refined grain intakes and serum C-reactive protein

2021 ◽  
Author(s):  
Riikka E. Taskinen ◽  
Sari Hantunen ◽  
Tomi-Pekka Tuomainen ◽  
Jyrki K. Virtanen
Keyword(s):  
Disease Risk ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Whole Grains ◽  
Dietary Factors ◽  
Low Grade ◽  
Dietary Intakes ◽  
Whole Grain ◽  
Refined Grains ◽  
Hs Crp

Abstract Background/objectives Epidemiological studies suggest that whole grain intake has inverse associations with low-grade inflammation, but findings regarding refined grains are inconclusive. Our objective was to investigate whether consumption of whole or refined grains is associated with serum high sensitivity CRP (hs-CRP). Subjects/methods The study included 756 generally healthy men and women aged 53–73 years from the Kuopio Ischaemic Heart Disease Risk Factory Study, examined in 1999–2001. Dietary intakes were assessed using 4-day food records. ANCOVA and linear regression were used for analyses. Results The mean intake of whole and refined grains was 136 g/day (SD 80) and 84 g/day (SD 46), respectively. Higher whole grain intake was associated with lower hs-CRP concentration and higher refined grain intake with higher concentration after adjustment for lifestyle and dietary factors. Each 50 g/d higher whole grain intake was associated with 0.12 mg/L (95% Cl 0.02–0.21 mg/L) lower hs-CRP concentration and each 50 g/d higher refined grain intake with 0.23 mg/L (95% Cl 0.08–0.38) higher concentration. Adjustment for fibre from grains attenuated the associations especially with whole grains. There were no statistically significant interactions according to gender or BMI (P for interactions >0.065). Conclusions The results of this study suggest that higher intake of whole grains is associated with lower concentrations of hs-CRP and higher intake of refined grains is associated with higher concentrations. However, especially the association with whole grain intake was attenuated after adjusting for fibre intake from grains, suggesting that cereal fibre may partly explain the association.

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