Homogeneous phase pyrophosphate (PPi) measurement (H3PIM) A non-radioactive, quantitative detection system for nucleic acid specific hybridization methodologies including gene amplification

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

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A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Biosensors ◽

10.3390/bios11120479 ◽

2021 ◽

Vol 11 (12) ◽

pp. 479

Author(s):

Soumi Sukla ◽

Prasenjit Mondal ◽

Subhajit Biswas ◽

Surajit Ghosh

Keyword(s):

Nucleic Acid ◽

Dengue Virus ◽

Magnetic Beads ◽

Molecular Beacon ◽

Detection System ◽

Denv Infection ◽

Molecular Beacons ◽

Nucleic Acid Amplification ◽

Complementary Sequence ◽

Reverse Transcription Pcr

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

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Evaluation of nucleic acid sequence based amplification using fluorescence resonance energy transfer (FRET-NASBA) in quantitative detection ofAspergillus18S rRNA

Medical Mycology ◽

10.3109/13693786.2010.507604 ◽

2011 ◽

Vol 49 (1) ◽

pp. 73-79 ◽

Cited By ~ 8

Author(s):

Chulmin Park ◽

Eun-Young Kwon ◽

Na-Young Shin ◽

Su-Mi Choi ◽

Si-Hyun Kim ◽

...

Keyword(s):

Energy Transfer ◽

Nucleic Acid ◽

Fluorescence Resonance Energy Transfer ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Nucleic Acid Sequence ◽

Quantitative Detection ◽

Fluorescence Resonance

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Design and Implementation of Experimental Consumable Management Device for the Automatic Nucleic Acid Detection System

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2016.13033 ◽

2016 ◽

Vol 16 (7) ◽

pp. 7069-7076

Author(s):

Jun Yu ◽

Zhu Chen ◽

Chao Wang ◽

Yana Hu ◽

Hongming Dong ◽

...

Keyword(s):

Nucleic Acid ◽

Detection System ◽

Nucleic Acid Detection ◽

Design And Implementation

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Abstract 5123: Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay

10.1158/1538-7445.sabcs18-5123 ◽

2019 ◽

Author(s):

Ru Cao ◽

Kris Lea ◽

Madhu Jasti ◽

Jeff Schageman ◽

Khalid Hanif ◽

...

Keyword(s):

Nucleic Acid ◽

Gene Amplification ◽

Cancer Cell ◽

Liquid Biopsy ◽

Genetic Mutation ◽

Biopsy Specimens ◽

Mutation Spectra ◽

Pan Cancer

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Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA

Clinical Chemistry ◽

10.1093/clinchem/45.11.1932 ◽

1999 ◽

Vol 45 (11) ◽

pp. 1932-1937 ◽

Cited By ~ 65

Author(s):

Andreas Nitsche ◽

Nina Steuer ◽

Christian Andreas Schmidt ◽

Olfert Landt ◽

Wolfgang Siegert

Keyword(s):

Real Time ◽

Human Cytomegalovirus ◽

Real Time Pcr ◽

Detection System ◽

Quantitative Detection ◽

Fluorescence Signal ◽

Hybridization Probe ◽

Sequence Detection ◽

Hybridization Probes ◽

Assay Time

Abstract Background: The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. Methods: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 101 to 107 HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.

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Ultraviolet Spectral Analysis and Quantitative Detection of Heptafluoroisobutyronitrile (C4F7N) in a C4F7N–Carbon Dioxide (CO2) Gas Mixture

Applied Spectroscopy ◽

10.1177/0003702819833702 ◽

2019 ◽

Vol 73 (8) ◽

pp. 917-926 ◽

Cited By ~ 3

Author(s):

Yin Zhang ◽

Xiaoxing Zhang ◽

Chang Liu ◽

Yi Li ◽

Zhaolun Cui ◽

...

Keyword(s):

Carbon Dioxide ◽

Absorption Spectroscopy ◽

Density Functional ◽

Sulfur Hexafluoride ◽

Detection System ◽

Spectral Characteristics ◽

Uv Absorption ◽

Quantitative Detection ◽

Gas Mixture ◽

Uv Absorption Spectroscopy

As an alternative to sulfur hexafluoride (SF6) with great potential for application, heptafluoroisobutyronitrile–carbon dioxide (C4F7N–CO2) gas mixture has been applied in various gas-insulated equipment. The insulation performance of the gas mixture is closely related to the mixing ratio. Therefore, accurate quantification of C4F7N in the C4F7N–CO2 gas mixture has very important engineering significance. At present, there are few reports on the rapid quantitative detection of the concentration of C4F7N in the gas mixture. In this paper, a rapid analytical method for C4F7N concentration based on ultraviolet (UV) absorption spectroscopy is constructed. The UV spectral characteristics of C4F7N molecules are calculated by density functional theory. The appropriate bands that can be detected are determined by analyzing the calculated results. A concentration detection system of C4F7N based on UV absorption spectroscopy is built. Through analysis of the calculated results and experimental results, a quantitative detection method of C4F7N in the C4F7N/CO2 gas mixture is determined. The method can achieve accurate detection of the concentration of the gas mixture in the conventional application range (including 4–10% C4F7N). The coefficient of the determination R2 of the concentration inversion curve reaches 0.999 and the inversion error ratio does not exceed 5%. The related research results provide an important reference for the engineering application of the gas mixture.

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