scholarly journals Programmable Hybridization Assemble Nicked Displacement Amplification for Detecting Ricin Toxin

2021 ◽  
Author(s):  
Yu Wang ◽  
Yuan Peng ◽  
Jialei Bai ◽  
Shuang Li ◽  
Dianpeng Han ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Isothermal Amplification ◽  
Design Rule ◽  
Hybridization Chain Reaction ◽  
Target Sequence ◽  
Toxic Substances ◽  
Trace Detection ◽  
Strand Displacement Amplification ◽  
Amplification Method ◽  
Universal Applicability

Abstract Isothermal amplification, such as hybridization chain reaction (HCR), is a simple and reliable method for detecting signal amplification. However, the hairpin in HCR will not fully participate in the reaction. And after the hairpin is opened, the distance between the fluorophore and the quencher does not change much. Therefore, the signal magnification is limited. Here, we designed a new isothermal amplification method named hybridization assemble nicked displacement amplification (HANDA), combining HCR and strand displacement amplification (SDA) ingeniously. HANDA first triggers HCR through the target sequence to form long double-strand DNA (dsDNA) with gaps. Then SDA is performed from the gap to obtain a large amount of single-stranded DNA (ssDNA), so as to achieve the purpose of double signal amplification. The base sequence of DNA hairpin had also been optimized. The best sequence design rule was found and had universal applicability. We have demonstrated that HANDA combined with DNA barcodes can be used for trace detection of ricin. This new isothermal amplification method provides an effective and universal platform for the trace detection of various toxic substances.

scholarly journals Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

Sensors ◽  
2021 ◽  
Vol 21 (2) ◽  
pp. 602
Author(s):  
Sandra Leonardo ◽  
Anna Toldrà ◽  
Mònica Campàs
Keyword(s):  
Food Safety ◽  
Environmental Monitoring ◽  
Rolling Circle Amplification ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
In Situ Monitoring ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Bacterial Detection ◽  
Efficient Detection

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

scholarly journals SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification

Micromachines ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 197
Author(s):  
Meiqing Liu ◽  
Haoran Li ◽  
Yanwei Jia ◽  
Pui-In Mak ◽  
Rui P. Martins
Keyword(s):  
Signal Amplification ◽  
Incubation Temperature ◽  
Dna Probes ◽  
Detection Sensitivity ◽  
N Gene ◽  
Rna Detection ◽  
Gold Standard Method ◽  
Complementary Method ◽  
Amplification Method ◽  
Virus Rna

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.

scholarly journals P02.06 A Novel Loop-Mediated Isothermal Amplification Method for Robust Detection of EGFR Mutations

2021 ◽  
Vol 16 (3) ◽  
pp. S248-S249
Author(s):  
Y. Saito ◽  
A. Matsui ◽  
S. Michiyuki ◽  
Y. Yamauchi ◽  
N. Takahashi ◽  
...  
Keyword(s):  
Isothermal Amplification ◽  
Egfr Mutations ◽  
Robust Detection ◽  
Loop Mediated Isothermal Amplification ◽  
Amplification Method

scholarly journals Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

2004 ◽  
Vol 70 (1) ◽  
pp. 621-624 ◽  
Author(s):  
Ram Savan ◽  
Arisa Igarashi ◽  
Satoru Matsuoka ◽  
Masahiro Sakai
Keyword(s):  
Rapid Detection ◽  
Japanese Flounder ◽  
Isothermal Amplification ◽  
Edwardsiella Tarda ◽  
Fish Disease ◽  
Fish Pathogen ◽  
Loop Mediated Isothermal Amplification ◽  
Diagnostic Protocol ◽  
Amplification Method ◽  
Hemolysin Gene

ABSTRACT Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.

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