scholarly journals Schnellnachweis von SARS-CoV-2 mit recombinase polymerase amplification

BIOspektrum ◽  
2020 ◽  
Vol 26 (6) ◽  
pp. 624-627
Author(s):  
Ole Behrmann ◽  
Iris Bachmann ◽  
Frank Hufert ◽  
Gregory Dame
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Dna Polymerase ◽  
Fluorescent Probes ◽  
Reverse Transcription ◽  
Nucleic Acid Detection ◽  
Detection Scheme ◽  
Recombination Proteins ◽  
Target Dna ◽  
Rapid Amplification

Abstract The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

scholarly journals Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽  
2007 ◽  
Vol 2 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Koray Ergunay ◽  
Gulcin Altinok ◽  
Bora Gurel ◽  
Ahmet Pinar ◽  
Arzu Sungur ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
San Francisco ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Parvovirus B19 ◽  
Etiologic Agent ◽  
Hydrops Fetalis ◽  
Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

Ribonuclease H-cleavable and recombinase-quenching fluorescent probes for the real-time detection of strand invasion based amplification

2019 ◽  
Vol 11 (43) ◽  
pp. 5568-5576
Author(s):  
Sonja Elf ◽  
Kevin E. Eboigbodin
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Real Time ◽  
Fluorescent Probes ◽  
Nucleic Acid Amplification ◽  
Ribonuclease H ◽  
Chain Reaction ◽  
Amplification Method ◽  
Polymerase Chain ◽  
Strand Invasion

SIBA is an established nucleic acid amplification method that is used as an alternative to polymerase chain reaction (PCR).

Colorimetric Nucleic Acid Detection Based on Gold Nanoparticles with Branched DNA

NANO ◽  
2020 ◽  
Vol 15 (08) ◽  
pp. 2050110
Author(s):  
Zhikun Zhang ◽  
Xiaojie Ye ◽  
Qingqing Liu ◽  
Cuixia Hu ◽  
Jimmy Yun ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Nucleic Acid ◽  
Genetic Diseases ◽  
Colorimetric Detection ◽  
Nucleic Acid Detection ◽  
Great Promise ◽  
Dna Nanostructures ◽  
Practical Applications ◽  
Branched Dna ◽  
Target Dna

Nucleic acid detection is becoming increasingly important in the diagnostics of genetic diseases for biological analysis. We herein propose gold nanoparticles as probe for colorimetric detection of nucleic acids with branched DNA nanostructures, which enables a novel and simple colorimetric biosensor. In our system, the target DNA specifically triggered two short-chain ssDNA probes to generate branched DNA nanostructures (Y-shape DNA), which prevent AuNPs from aggregation in aqueous NaCl solution. On the contrary, when the target DNA did not exist, gold nanoparticles were unstable and aggregated easily because there is no anti-aggregation function from Y-shape DNA. Sensor response was found to be proportional to the target DNA concentration from 5 to 100[Formula: see text]nM, with detection limits determined as 5[Formula: see text]nM. The developed platform is for colorimetric nucleic acid detection without enzymes, label and modification holds great promise for practical applications.

scholarly journals Impact of COVID-19 pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results

2021 ◽  
pp. 1-18
Author(s):  
Jonathan B. Gubbay ◽  
Heather Rilkoff ◽  
Heather L. Kristjanson ◽  
Jessica D. Forbes ◽  
Michelle Murti ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Real Time ◽  
Reverse Transcription ◽  
Predictive Value ◽  
Large Scale ◽  
Rdrp Gene ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Test Probability

Abstract Objectives Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent presymptomatic or asymptomatic infection, resolved infection with persistent RNA shedding, or a false positive test. This study assessed positive predictive value of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay. Methods A total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. Results Significantly fewer (15.6%) positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤ 3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in each of the four groups with higher pre-test probability (individual group range 50·0% to 85·0%). Conclusions Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level.

scholarly journals Chloro-Substituted Naphthyridine Derivative and Its Conjugate with Thiazole Orange for Highly Selective Fluorescence Sensing of an Orphan Cytosine in the AP Site-Containing Duplexes

2020 ◽  
Vol 10 (12) ◽  
pp. 4133
Author(s):  
Chun-xia Wang ◽  
Yusuke Sato ◽  
Takashi Sugimoto ◽  
Norio Teramae ◽  
Seiichi Nishizawa
Keyword(s):  
Nucleic Acid ◽  
Sequence Analysis ◽  
Fluorescent Probes ◽  
Dna Duplexes ◽  
Thiazole Orange ◽  
Fluorescence Sensing ◽  
Binding Selectivity ◽  
Target Dna ◽  
Fluorescence Signaling ◽  
Ap Site

Fluorescent probes with the binding selectivity to specific structures in DNAs or RNAs have gained much attention as useful tools for the study of nucleic acid functions. Here, chloro-substituted 2-amino-5,7-dimethyl-1,8-naphthyridine (ClNaph) was developed as a strong and highly selective binder for target orphan cytosine opposite an abasic (AP) site in the DNA duplexes. ClNaph was then conjugated with thiazole orange (TO) via an alkyl spacer (ClNaph–TO) to design a light-up probe for the detection of cytosine-related mutations in target DNA. In addition, we found the useful binding and fluorescence signaling of the ClNaph–TO conjugate to target C in AP site-containing DNA/RNA hybrid duplexes with a view toward sequence analysis of microRNAs.

scholarly journals Simultaneous Absolute Quantification of Target and Control Templates by Real-Time Fluorescence Reverse Transcription-PCR Using 4-(4′-Dimethylaminophenylazo)benzoic Acid as a Dark Quencher Dye

2001 ◽  
Vol 47 (3) ◽  
pp. 486-490 ◽  
Author(s):  
Karl-Anton Kreuzer ◽  
Alexander Bohn ◽  
Joachim Lupberger ◽  
Jerome Solassol ◽  
Philipp le Coutre ◽  
...  
Keyword(s):  
Benzoic Acid ◽  
Real Time ◽  
Correlation Coefficient ◽  
Fluorescent Probes ◽  
Reverse Transcription ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Rt Pcr ◽  
Internal Reference ◽  
Reverse Transcription Pcr

Abstract Background: Despite the many advantages of real-time fluorescence reverse transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template. Methods: For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrument. Fluorescent probes were labeled with either 6-carboxy-fluorescein or hexachloro-6-carboxy-fluorescein as reporter dye and 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sensitivity and specificity of this assay, serial dilutions of reference and target templates were analyzed in one PCR reaction. In the presence of 10 β-actin molecules as control templates, 105 bcr/abl molecules were amplified, and 105 β-actin molecules were amplified in the presence of 10 bcr/abl copies. We also performed single and duplex measurements on samples from five patients with documented Philadelphia chromosome-positive chronic myelogenous leukemia disease courses (72 samples) and three with minor bcr/abl+ acute myelogenous leukemias (26 samples). Results: For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for starting template amounts and threshold cycle values was 0.99; for m-bcr/abl, r = 0.96, indicating a precise log-linear relation for 10–105 copies/100 ng of cDNA. In the same PCR reactions, r = 0.99 for β-actin (coamplified with M-bcr/abl or m-bcr/abl) for 103–107 copies/100 ng cDNA. The linear correlation coefficient for single and duplex measurements was 0.98 for M- and m-bcr/abl in patient samples. Conclusions: DABCYL can be used as dark quencher fluorophore in real-time fluorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and β-actin transcripts allows monitoring of bcr/abl+ leukemias.

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