DETECTION AND APPLICATION OF MICROFLUIDIC ISOTHERMAL AMPLIFICATION ON CHIP

2008 ◽  
Vol 01 (02) ◽  
pp. 257-265 ◽  
Author(s):  
GUOLIANG HUANG ◽  
XIAOYONG YANG ◽  
JIANG ZHU ◽  
SHUKUAN XU ◽  
CHENG DENG ◽  
...  
Keyword(s):  
Detection System ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Amplification Method ◽  
Amplification Process ◽  
Different Temperatures ◽  
System Temperature ◽  
Polymerase Chain ◽  
On Chip

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method. Compared with the widely utilized polymerase chain reaction (PCR), LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different temperatures. In this paper, we designed and fabricated different kinds of polycarbonate (PC) microfluid chips, explored appropriate reaction condition for LAMP in microenvironment (1 nL → 10 μL), and developed a microfluidic isothermal amplification detection system. The DNA optimal amplification temperature is obtained; the starting time of exponential amplification of DNA is put forward farther. The optimal condition of DNA amplification in microenvironment, with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.

scholarly journals Detection of SARS-CoV-2 RNA by a Multiplex Reverse-Transcription Loop-Mediated Isothermal Amplification Coupled with Melting Curves Analysis

2021 ◽  
Vol 22 (11) ◽  
pp. 5743
Author(s):  
Igor P. Oscorbin ◽  
Georgiy Yu. Shevelev ◽  
Ksenia A. Pronyaeva ◽  
Andrey A. Stepanov ◽  
Darya V. Shamovskaya ◽  
...  
Keyword(s):  
Internal Control ◽  
Reverse Transcription ◽  
Control Sample ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Single Tube ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Process

Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients’ nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.

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).

scholarly journals IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Martin Jensen Søe ◽  
Mikkel Rohde ◽  
Jens Mikkelsen ◽  
Peter Warthoe
Keyword(s):  
Nucleic Acid ◽  
Candida Glabrata ◽  
Simultaneous Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

scholarly journals Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

2020 ◽  
Author(s):  
Pierre Garneret ◽  
Etienne Coz ◽  
Elian Martin ◽  
Jean-Claude Manuguerra ◽  
Elodie Brient-Litzler ◽  
...  
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Rna Extraction ◽  
Dna Amplification ◽  
Developed Countries ◽  
Temperature Cycling ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Molecular Testing

In order to respond to the urgent request of massive testing, developed countries perform nucleic acid amplification tests (NAAT) of SARS-CoV-2 in centralized laboratories. Real-time RT - PCR (Reverse transcription - Polymerase Chain Reaction) is used to amplify the viral RNA and enable its detection. Although PCR is 37 years old, it is still considered, without dispute, as the gold standard. PCR is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings. In the present work, by harnessing progress made in the past two decades in DNA amplification, microfluidics and membrane technologies, we succeeded to create a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT - LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or highly fluorescent probes. Depending on the viral load, the detection takes between twenty minutes and one hour. Using pools of naso-pharyngal clinical samples, we estimated a sensitivity comparable to RT-qPCR (up to a Cycle threshold of 39, equivalent to <0.1 TCID50 per mL) and a 100% specificity, for other human coronaviruses and eight respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called COVIDISC to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment paves the way towards a large dissemination of this device. The perspective of a reliable SARS-CoV-2 point of care detection, highly performing, that would deliver on-site results in less than one hour opens up a new efficient approach to manage the pandemics.

Shortening distance of forward and reverse primers for nucleic acid isothermal amplification

2014 ◽  
Vol 395 (6) ◽  
pp. 679-684
Author(s):  
Qu Haitao ◽  
Zhang Wenchao ◽  
Zhang Xiaohui ◽  
Wang Xiujun ◽  
Li Sulong
Keyword(s):  
Nucleic Acid ◽  
Reaction Times ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Amplification Efficiency ◽  
Specific Method ◽  
Detection Techniques ◽  
Amplification Method ◽  
Reverse Primer ◽  
Short Time

Abstract Existent nucleic acid isothermal detection techniques for clinical diseases are difficult to promote greatly due to limitations in such aspects as methodology, costs of detection, amplification efficiency and conditions for operation. There is therefore an urgent need for a new isothermal amplification method with the characteristics of high accuracy, easy operation, short time of detection and low costs. We have devised a new method of nucleic acid isothermal amplification using Bst DNA polymerase under isothermal conditions (60–65°C). We call this method of amplification by shortening the distance between forward and reverse primers for nucleic acid isothermal amplification SDAMP. The results demonstrated that this technique is highly sensitive, specific and has short reaction times (40–60 min). Results of sequencing show that the products of SDAMP amplification are mainly polymers formed by series connection of monomers formed through linkage of forward primer and complementary sequences in reverse primer via a few bases. The method is different from current methods of nucleic acid amplification. Our study shows, however, that it is a specific method of nucleic acid isothermal amplification depending on interactions between primers and DNA template.

scholarly journals Variability of Potato virus Y in Tomato Crops in Poland and Development of a Reverse-Transcription Loop-Mediated Isothermal Amplification Method for Virus Detection

2015 ◽  
Vol 105 (9) ◽  
pp. 1270-1276 ◽  
Author(s):  
Beata Hasiów-Jaroszewska ◽  
Joanna Stachecka ◽  
Julia Minicka ◽  
Mateusz Sowiński ◽  
Natasza Borodynko
Keyword(s):  
Potato Virus ◽  
Reverse Transcription ◽  
Potato Virus Y ◽  
Protein Gene ◽  
Virus Detection ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method ◽  
Polymerase Chain

A collection of 147 Potato virus Y (PVY) isolates from tomato, originating from several commercial fields and greenhouses in different regions of Poland, was tested for the presence of PVY by reverse-transcription polymerase chain reaction. However, in some cases, the results obtained were ambiguous. Therefore, a sensitive reverse-transcription loop-mediated isothermal amplification method was developed for rapid detection of PVY isolates. Phylogenetic and recombination analyses were performed based on sequences of the coat protein gene. In comparison with results obtained in 2008, the presence of other strains besides PVYNWi-P was confirmed. A novel recombinant between PVYNTN and PVYNWi-P strains was detected. Our results indicate an increasing distribution and variability of the PVY population on tomato in Poland.

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