scholarly journals A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples

2016 ◽  
Vol 162 (2) ◽  
pp. 477-486 ◽  
Author(s):  
C. Claus ◽  
S. Bergs ◽  
N. C. Emmrich ◽  
J. M. Hübschen ◽  
A. Mankertz ◽  
...  
Keyword(s):  
Rubella Virus ◽  
Clinical Samples ◽  
One Step

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Rapid electrochemical detection of coronavirus SARS-CoV-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Thanyarat Chaibun ◽  
Jiratchaya Puenpa ◽  
Tatchanun Ngamdee ◽  
Nimaradee Boonapatcharoen ◽  
Pornpat Athamanolap ◽  
...  
Keyword(s):  
Electrochemical Biosensor ◽  
Rolling Circle Amplification ◽  
Clinical Samples ◽  
Rolling Circle ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Sandwich Hybridization ◽  
Redox Active ◽  
One Step ◽  
The One

AbstractCoronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

scholarly journals Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution

2002 ◽  
Vol 48 (2) ◽  
pp. 269-277 ◽  
Author(s):  
Piia Tarkkinen ◽  
Tom Palenius ◽  
Timo Lövgren
Keyword(s):  
Whole Blood ◽  
Solid Phase ◽  
Point Of Care ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Cardiac Complications ◽  
Fluorescence Signal ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
One Step

Abstract Background: Recently, measurement of very low concentrations of C-reactive protein (CRP) has gained popularity as a potential new means for predicting the risk of future cardiac complications. In this study, we demonstrate the feasibility of a kinetic, one-step microparticle assay for quantitative determination of extremely low and high CRP concentrations in the limited timeframe typical for point-of-care testing. Methods: A noncompetitive, kinetic CRP immunoassay was developed that uses individual, porous microparticles as the solid phase. The microparticles were covalently coated with a monoclonal capture antibody, and the monoclonal detection antibody was labeled with europium. The one-step binding reaction was stopped by washing after 2 min of incubation, and the fluorescence signal of individual particles was measured. Results: The analytical detection limit (mean of zero calibrator + 3 SD) was 0.00016 mg/L CRP. Clinical samples were diluted 400-fold before assay to cover the CRP concentration range of 0.064–1200 mg/L. The assay correlated well with the Dade Behring N High Sensitivity CRP assay (for 0–10 mg/L, r = 0.969, Sy|x = 0.68, n = 54; for 0–350 mg/L, r = 0.969, Sy|x = 11.7, n = 100). The within- and between-run CVs based on calculated concentrations were, respectively, 9–16% and 14% at 0.11 mg/L, 4.5–12% and 8.2% at 4.2 mg/L, and 3.5–6.3% and 4.4% at 105 mg/L, with a CV <15% at 0.2 mg/L and above. Conclusions: Use of the kinetic microparticle approach combined with time-resolved fluorometry allows ultrasensitive quantification of CRP in whole blood in 2 min with a linear assay range spanning more than four orders of magnitude.

A micro tissue culture test for the titration and neutralization of rubella virus

1969 ◽  
Vol 15 (1) ◽  
pp. 67-71 ◽  
Author(s):  
John Furesz ◽  
Pierre Moreau ◽  
Walter Yarosh
Keyword(s):  
Tissue Culture ◽  
Rubella Virus ◽  
Fetal Calf Serum ◽  
Constant Pressure ◽  
Calf Serum ◽  
Rabbit Kidney ◽  
Tissue Cultures ◽  
Virus Titration ◽  
One Step ◽  
Microneutralization Test

A simple and reproducible micro tissue culture assay has been devised in RK13 and LLC-RK1 rabbit kidney cells for the titration and neutralization of rubella virus. In this "one-step" assay all virus and serum dilutions were prepared with spiral loops in disposable microplates and tissue cultures suspended in medium 199 and 3% horse or fetal calf serum were added to the microcups simultaneously. Micro tissue cultures were kept in a humidified incubator (36 °C) under a constant pressure of 5% CO2 for 8 days and were read microscopically for viral cytopathic changes on the seventh and eighth day. The microneutralization test performed in LLC-RK1 cell cultures was shown to be a reliable method for the detection of small amounts of rubella antibodies in human sera.The micro assay may be also applied to the virus titration of live, attenuated rubella vaccines.

Assessment of a One-Step Intracellular Staining in Th1, Th2 and Th17 Cells of Clinical Samples

2015 ◽  
Vol 61 (05+06/2015) ◽  
Author(s):  
Xianghua Lin ◽  
Xiaohong Luo ◽  
Ying Wang ◽  
Qiuju Wu ◽  
Wangxian Xiao ◽  
...  
Keyword(s):  
Th17 Cells ◽  
Clinical Samples ◽  
Intracellular Staining ◽  
One Step

scholarly journals One-step simultaneous detection ofUreaplasma parvumand genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology

2017 ◽  
Vol 65 (2) ◽  
pp. 153-158 ◽  
Author(s):  
M.S. Payne ◽  
L.L. Furfaro ◽  
R. Tucker ◽  
L.Y. Tan ◽  
E. Mokany
Keyword(s):  
Simultaneous Detection ◽  
Clinical Samples ◽  
One Step

scholarly journals Rapid Detection and Molecular Typing of Dengue Virus by Using Multiplex-Nested-RT-PCR

2015 ◽  
Vol 11 (2) ◽  
Author(s):  
Nastiti Wijayanti ◽  
Hera Nirwati ◽  
Tri Wibawa ◽  
Aris Haryanto ◽  
S. Sutaryo
Keyword(s):  
Dengue Virus ◽  
Nested Pcr ◽  
Genotypic Variation ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Virus Serotype ◽  
Pcr Multiplex ◽  
Pcr Products ◽  
One Step ◽  
Nested Pcr Assays

world. We have evaluated the combination of one-step RT-PCR and multiplex nested PCR assays for detectingdengue viruses from clinical samples. Twelve patients were screened for the dengue virus, using a pair of primersthat conserve for several Flavivirus. The results showed that in 12 suspect patients, 100% were positive for Flavivirusand there are some genotypic variation among them, that indicated by several RT-PCR products higher than 511 bp,the expected product for RT-PCR. Further assay was performed to clarify the presence and serotypes of dengue virususing multiplex nested PCR. Serotyping results indicated that 83,3% of samples can be confirmed for dengue virus.Among the dengue virus positive 16,7 % are dengue-2, 16.7 % are dengue-3, and the most common 50% are dengue-4,whereas dengue-1 were not found among the patients. The combination of RT-PCR and multiplex nested PCR assaycan be used for rapid analysis dengue samples in early phase which is potentially useful for clinical, epidemiologyand also evolutionary studies.Key words: Flavivirus, dengue virus, serotype, RT-PCR, multiplex nested PCR

scholarly journals One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

2021 ◽  
Vol 12 ◽  
Author(s):  
Jia Xie ◽  
Xiaohan Yang ◽  
Lei Duan ◽  
Keyi Chen ◽  
Pan Liu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Effective Vaccine ◽  
Lateral Flow Strip ◽  
Coxsackievirus A6 ◽  
One Step

Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.

Sign in / Sign up

Export Citation Format

Share Document