scholarly journals Standardization of an LNA-based TaqMan assay qPCR analysis for Aspiculuris tetraptera DNA in mouse faeces

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Keishiro Isayama ◽  
Kenji Watanabe ◽  
Mariko Okamoto ◽  
Tomoaki Murata ◽  
Yoichi Mizukami
Keyword(s):  
Genomic Dna ◽  
High Sensitivity ◽  
Locked Nucleic Acid ◽  
Detection Sensitivity ◽  
Taqman Assay ◽  
Highly Sensitive ◽  
Aspiculuris Tetraptera ◽  
Pcr Techniques ◽  
Quantitative Pcr Assay ◽  
Direct Microscopic Observation

Abstract Background Aspiculuris tetraptera, as a parasitic pinworm, is most frequently detected in laboratory mice, and transmission is mediated by the eggs contained in the faeces of infected mice. A highly sensitive and quantitative faeces-based diagnostic tool would be useful for the early detection of A. tetraptera to inhibit the expansion of infection. In this study, we developed a quantitative assay that exhibits high sensitivity in detecting A. tetraptera in faeces using PCR techniques. Results Endpoint PCR demonstrated the detection of A. tetraptera DNA in 0.5 ng genomic DNA extracted from the faeces of infected mice. To quantitatively detect the small amount of A. tetraptera DNA, locked nucleic acid (LNA)-based primers and LNA-based TaqMan probes were used for the quantitative PCR assay (qPCR). The combination of LNA-based DNA increased detection sensitivity by more than 100-fold compared to using normal oligo DNAs. The copy number of the A. tetraptera DNA detected was positively related to the infected faeces-derived genomic DNA with a simple linearity regression in the range of 20 pg to 15 ng of the genomic DNA. To more conveniently detect infection using faeces, the LNA-based TaqMan assay was applied to the crude fraction of the faeces without DNA purification. An assay using ethanol precipitation of the faeces yielded results consistent with those of direct microscopic observation. Conclusion The LNA-TaqMan assay developed in this study quantitatively detects A. tetraptera infection in mouse faeces.

Amplification-Free DNA Detection Using a Microtip-Sensor Decorated With LNA Probes for Rapid TB Screening

2011 ◽  
Author(s):  
Shinnosuke Inoue ◽  
Woon-Hong Yeo ◽  
Jong-Hoon Kim ◽  
Jae-Hyun Chung ◽  
Kyong-Hoon Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Staphylococcus Epidermidis ◽  
Genomic Dna ◽  
Bacterial Culture ◽  
Low Cost ◽  
Surrogate Marker ◽  
High Sensitivity ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Highly Sensitive

Tuberculosis (TB) is an epidemic affecting one-third of the world’s population, mostly in developing and low-resource settings. People having active pulmonary TB are considered highly infectious; therefore, it is critical to identify and treat these patients rapidly before spreading to others. However, the most reliable TB diagnostic methods of bacterial culture or nucleic acid amplification are time-consuming and expensive. The challenge of TB diagnosis lies in highly sensitive and specific screening with low cost. Here, we present an LNA-modified microtip-sensor, which is capable of selectively detecting low-abundance DNA from bacteria. When genomic DNA of Bacillus Calmette-Gue´rin (BCG, a surrogate marker of Mycobacterium bovis), and genomic DNA of Staphylococcus epidermidis (S. epi) are used, the microtip-sensor yields the detection limit of 1,000 copies/mL within 20 minutes. The high sensitivity and specificity approaching nucleic acid amplification methods can potentially overcome the current challenges for rapid TB screening.

scholarly journals Pan-Piscine Orthoreovirus (PRV) Detection Using Reverse Transcription Quantitative PCR

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1548
Author(s):  
Julie Zhao ◽  
Niccolò Vendramin ◽  
Argelia Cuenca ◽  
Mark Polinski ◽  
Laura M. Hawley ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
High Sensitivity ◽  
Test Results ◽  
Reverse Transcription Quantitative Pcr ◽  
Highly Sensitive ◽  
Surveillance Programs ◽  
America South ◽  
Universal Detection ◽  
Quantitative Pcr Assay

Piscine orthoreovirus (PRV) infects farmed and wild salmon and trout species in North America, South America, Europe, and East Asia. PRV groups into three distinct genotypes (PRV-1, PRV-2, and PRV-3) that can vary in distribution, host specificity, and/or disease potential. Detection of the virus is currently restricted to genotype specific assays such that surveillance programs require the use of three assays to ensure universal detection of PRV. Consequently, herein, we developed, optimized, and validated a real-time reverse transcription quantitative PCR assay (RT-qPCR) that can detect all known PRV genotypes with high sensitivity and specificity. Targeting a conserved region at the 5′ terminus of the M2 segment, the pan-PRV assay reliably detected all PRV genotypes with as few as five copies of RNA. The assay exclusively amplifies PRV and does not cross-react with other salmonid viruses or salmonid host genomes and can be performed as either a one- or two-step RT-qPCR. The assay is highly reproducible and robust, showing 100% agreement in test results from an inter-laboratory comparison between two laboratories in two countries. Overall, as the assay provides a single test to achieve highly sensitive pan-specific PRV detection, it is suitable for research, diagnostic, and surveillance purposes.

scholarly journals Highly Sensitive, Selective, Flexible and Scalable Room-Temperature NO2 Gas Sensor Based on Hollow SnO2/ZnO Nanofibers

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6475
Author(s):  
Jiahui Guo ◽  
Weiwei Li ◽  
Xuanliang Zhao ◽  
Haowen Hu ◽  
Min Wang ◽  
...  
Keyword(s):  
Room Temperature ◽  
Gas Sensing ◽  
Positive Impact ◽  
High Sensitivity ◽  
Detection Sensitivity ◽  
Pure Sno2 ◽  
Flexible Devices ◽  
Low Concentrations ◽  
Highly Sensitive ◽  
Sensitivity And Selectivity

Semiconducting metal oxides can detect low concentrations of NO2 and other toxic gases, which have been widely investigated in the field of gas sensors. However, most studies on the gas sensing properties of these materials are carried out at high temperatures. In this work, Hollow SnO2 nanofibers were successfully synthesized by electrospinning and calcination, followed by surface modification using ZnO to improve the sensitivity of the SnO2 nanofibers sensor to NO2 gas. The gas sensing behavior of SnO2/ZnO sensors was then investigated at room temperature (~20 °C). The results showed that SnO2/ZnO nanocomposites exhibited high sensitivity and selectivity to 0.5 ppm of NO2 gas with a response value of 336%, which was much higher than that of pure SnO2 (13%). In addition to the increase in the specific surface area of SnO2/ZnO-3 compared with pure SnO2, it also had a positive impact on the detection sensitivity. This increase was attributed to the heterojunction effect and the selective NO2 physisorption sensing mechanism of SnO2/ZnO nanocomposites. In addition, patterned electrodes of silver paste were printed on different flexible substrates, such as paper, polyethylene terephthalate and polydimethylsiloxane using a facile screen-printing process. Silver electrodes were integrated with SnO2/ZnO into a flexible wearable sensor array, which could detect 0.1 ppm NO2 gas after 10,000 bending cycles. The findings of this study therefore open a general approach for the fabrication of flexible devices for gas detection applications.

scholarly journals Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization

2015 ◽  
Vol 21 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Won S. Hong ◽  
Hannah M. Pezzi ◽  
Andrea R. Schuster ◽  
Scott M. Berry ◽  
Kyung E. Sung ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Transforming Growth Factor ◽  
Culture Media ◽  
High Sensitivity ◽  
Transforming Growth Factor Β1 ◽  
Detection Sensitivity ◽  
Media Optimization ◽  
Sensitive Cell ◽  
Optimization Study ◽  
Highly Sensitive

Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron–like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell–derived neurons as well as the mouse lethality assay.

High-sensitivity detection of gold labels by high-angle dark-field STEM imaging

1990 ◽  
Vol 48 (3) ◽  
pp. 892-893 ◽  
Author(s):  
Max T. Otten
Keyword(s):  
High Sensitivity ◽  
Dark Field ◽  
Bright Field ◽  
Backscattered Electron Imaging ◽  
Backscattered Electrons ◽  
Average Atomic Number ◽  
Highly Sensitive ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
High Sensitivity Detection

Labelling of antibodies with small gold probes is a highly sensitive technique for detecting specific molecules in biological tissue. Larger gold probes are usually well visible in TEM or STEM Bright-Field images of unstained specimens. In stained specimens, however, the contrast of the stain is frequently the same as that of the gold labels, making it virtually impossible to identify the labels, especially when smaller gold labels are used to increase the sensitivity of the immunolabelling technique. TEM or STEM Dark-Field images fare no better (Figs. 1a and 2a), again because of the absence of a clear contrast difference between gold labels and stain.Potentially much more useful is backscattered-electron imaging, since this will show differences in average atomic number which are sufficiently large between the metallic gold and the stains normally used. However, for the thin specimens and at high accelerating voltages of the STEM, the yield of backscattered electrons is very small, resulting in a very weak signal. Consequently, the backscattered-electron signal is often too noisy for detecting small labels, even for large spot sizes.

scholarly journals Role of MRI evaluation in acute secondary inability to walk in children

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
K. H. Sedeek ◽  
K. Aboualfotouh ◽  
S. M. Hassanein ◽  
N. M. Osman ◽  
M. H. Shalaby
Keyword(s):  
Transverse Myelitis ◽  
High Sensitivity ◽  
Acute Disseminated Encephalomyelitis ◽  
Limb Weakness ◽  
Non Invasive ◽  
Highly Sensitive ◽  
Common Causes ◽  
Bilateral Lower Limb ◽  
Severity Of The Disease

Abstract Background Acute bilateral lower limb weakness is a common problem in children which necessitates a rapid method for diagnosis. MRI is a non-invasive imaging technique that produces high-quality images of the internal structure of the brain and spinal cord. Results MRI was very helpful in reaching rapid and prompt diagnosis in children with acute inability to walk. Acute disseminated encephalomyelitis (ADEM), Guillain–Barré syndrome (GBS), and acute transverse myelitis (ATM) were the most common causes in our study. MRI proved to be of high sensitivity in detecting the lesions and reaching the diagnosis in ADEM and GBS; however, there was no significant relation between the lesions’ size, enhancement pattern, and severity of the disease or prognosis, yet in ATM the site of the lesion and number of cord segment affection were significantly related to the severity of the disease and prognosis. Conclusion MRI is a quick tool to reach the diagnosis of children with acute secondary inability to walk, and to eliminate other differential diagnosis which is essential for proper treatment and rapid full recovery. It is highly sensitive in detecting the lesions, their site and size.

scholarly journals Highly sensitive detection of nitrobenzene by a series of fluorescent 2D zinc(ii) metal–organic frameworks with a flexible triangular ligand

RSC Advances ◽  
2021 ◽  
Vol 11 (39) ◽  
pp. 23975-23984
Author(s):  
Xue Yang ◽  
Yixia Ren ◽  
Hongmei Chai ◽  
Xiufang Hou ◽  
Zhixiang Wang ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Metal Organic Frameworks ◽  
High Sensitivity ◽  
Fluorescent Sensors ◽  
Sensitive Detection ◽  
Donor Ligands ◽  
Highly Sensitive ◽  
Metal Organic ◽  
Highly Sensitive Detection

Four fluorescent 2D Zn-MOFs based on a flexible triangular ligand and linear N-donor ligands are hydrothermally prepared and used to detect nitrobenzene in aqueous solution with high sensitivity, demonstrating their potential as fluorescent sensors.

Rapid, highly sensitive and quantitative detection of interleukin 6 based on SERS magnetic immunoassay

2021 ◽  
Vol 13 (15) ◽  
pp. 1823-1831
Author(s):  
Xiaomei Wang ◽  
Li Ma ◽  
Shijiao Sun ◽  
Tingwei Liu ◽  
Hao Zhou ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
High Sensitivity ◽  
Quantitative Detection ◽  
Detection Time ◽  
Highly Sensitive ◽  
Sandwich Method

We have developed a SERS magnetic immunoassay method based on the principle of sandwich method for rapid and quantitative detection of IL-6. The developed SERS method has the advantages of high sensitivity and detection time is only 15 min.

scholarly journals Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1207
Author(s):  
Hong Jae Cheon ◽  
Quynh Huong Nguyen ◽  
Moon Il Kim
Keyword(s):  
Magnetic Nanoparticles ◽  
High Sensitivity ◽  
High Specificity ◽  
Choline Oxidase ◽  
Fluorescent Detection ◽  
One Pot ◽  
Site Structure ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Peroxidase Mimics

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

scholarly journals Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lenka Ulrychová ◽  
Pavel Ostašov ◽  
Marta Chanová ◽  
Michael Mareš ◽  
Martin Horn ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Schistosoma Mansoni ◽  
Rna Sequencing ◽  
Serine Proteases ◽  
Expression Patterns ◽  
High Sensitivity ◽  
Host Parasite Interactions ◽  
Highly Sensitive ◽  
Host Parasite

Abstract Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphic abstract

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