Development of recombinase polymerase amplification combined with lateral flow detection assay for rapid and visual detection of Ralstonia solanacearum in tobacco

Plant Disease ◽  
2021 ◽  
Author(s):  
Changfeng Li ◽  
Yuliang Ju ◽  
Xun Wu ◽  
Pengfei Shen ◽  
Le Cao ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Visual Detection ◽  
High Sensitivity ◽  
High Specificity ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Molecular Diagnostic ◽  
Tobacco Stem ◽  
Detection Assay ◽  
Flow Detection

Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease that results in severe losses to tobacco (Nicotiana tabacum) production in China. In this study, a novel RPA-LFD assay for the rapid visual detection of R. solanacearum was established using recombinase polymerase amplification (RPA) and lateral-flow dipstick (LFD). The RPA-LFD assay was performed at 37°C in 30 min without complex equipment. Targeting the sequence of the RipTALI-9 gene, we designed RPA primers (Rs-rpa-F/R) and an LF probe (Rs-LF-probe) that showed high specificity to R. solanacearum. The sensitivity of RPA-LFD assay to R. solanacearum was the same as that in conventional PCR at 1 pg genomic DNA, 102 CFU/g artificially inoculated tobacco stem, and 103 CFU/g artificially inoculated soil. The RPA-LFD assay could also detect R. solanacearum from plant and soil samples collected from naturally infested tobacco fields. These results suggest that the RPA-LFD assay developed in this study is a rapid, accurate molecular diagnostic tool with high sensitivity for the detection of R. solanacearum.

scholarly journals Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Youngeun Kim ◽  
Adam B. Yaseen ◽  
Jocelyn Y. Kishi ◽  
Fan Hong ◽  
Sinem K. Saka ◽  
...  
Keyword(s):  
Visual Detection ◽  
High Specificity ◽  
Lateral Flow ◽  
Single Strand ◽  
Isothermal Amplification ◽  
Recombinase Polymerase Amplification ◽  
Sensitive Detection ◽  
Rna Detection ◽  
Flow Devices ◽  
Rapid Conversion

AbstractWe report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 µL reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

scholarly journals A Novel Rapid Visual Detection of Toxoplasma Gondii by Combining Recombinase Polymerase Amplification and Lateral Flow Dipstick Coupled With CRISPR-Cas13a Fluorescence Assay

2021 ◽  
Author(s):  
Jinhong Zhao ◽  
Yuanyuan Li ◽  
Qiqi Xue ◽  
Zhiwei Zhu ◽  
Minghui Zou ◽  
...  
Keyword(s):  
Visual Detection ◽  
High Specificity ◽  
Lateral Flow ◽  
Parasitic Disease ◽  
Blood Samples ◽  
Naked Eye ◽  
Detection Assay ◽  
Lateral Flow Dipstick ◽  
Sophisticated Equipment ◽  
Positive Rate

Abstract Background: Toxoplasmosis caused by infecting with Toxplasma gondii is a kind of parasitic disease that prevalent all over the world and does great harm to pregnant women and newborns. Effective, rapid and accurate diagnosis T. gondii is urgently needed to prevent and treatment the toxoplasmosis. The purpose of this study was to develop a rapid visual detection assay using recombinase aided amplification (RAA) and lateral flow dipstick (LFD) coupled with CRISPR-Cas13a fluorescence, henceforth RAA-Cas13a-LFD, for detection of T. gondii.Methods: Targeting 529bp gene of T. gondii, the primers and probes for RAA-Cas13a-LFD assay were designed and screened. The reaction time of RAA-LFD-Cas13a assay was optimized, as well as the sensitivity and specificity was further validated. Finally, the diagnostic performance of T. gondii was evaluated using the RAA-Cas13a-LFD assay for clinical blood samples.Results: The RAA-Cas13a-LFD assay was performed in an incubator block at 37℃ within 2h, and the amplicons were visible through LFD for naked eye visualization. The detection limit of the developed RAA-Cas13a-LFD assay was 1×10-6 ng/μL with high specificity for T. gondii. Compared with qPCR assay, there was a consistent positive rate among the clinical blood samples. Conclusion: In this study, A rapid and visual RAA-Cas13a-LFD assay was developed. It requires no sophisticated equipment and shows promise for on-site surveillance of T. gondii.

scholarly journals Engineered CRISPR/Cas12a Enables Rapid SARS-CoV-2 Detection

2020 ◽  
Author(s):  
Long T. Nguyen ◽  
Santosh R. Rananaware ◽  
Brianna L.M. Pizzano ◽  
Brandon T. Stone ◽  
Piyush K. Jain
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Single Copy ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Magnesium Concentration ◽  
Clinical Validation ◽  
Respiratory Pathogens ◽  
Reporter Assay ◽  
Wide Range

ABSTRACTThe coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies such as DETECTR, SHERLOCK, and STOPCovid have emerged as a rapid and affordable platform that can shape the future of diagnostics. Recently, we reported engineered crRNAs for Cas12a, called ENHANCE, that enables enhanced detection of nucleic acids. Here we report development, clinical validation, and advancement of ENHANCE platform for detecting SARS-CoV-2. With an RT-LAMP pre-amplification step, ENHANCE detects samples down to a single copy with 95% accuracy and shows high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. Utilizing LbCas12a-mediated trans-cleavage activity, ENHANCE works robustly in a wide range of magnesium concentration (3 mM-13 mM), allowing for further assay optimization. Additionally, ENHANCEv2 is developed to further improve the previously reported ENHANCE. ENHANCEv2 employs mutated LbCas12aD156R, engineered chimeric DNA-extended crRNA, and a dual reporter for both fluorescence-based reporter assay and lateral flow assay. Both ENHANCE and ENHANCEv2 are validated in 62 clinical nasopharyngeal swabs, showing 60/62 (96.7%) agreement with RT-qPCR results, and using only 5 μL of sample and 20 minutes of CRISPR reaction. Lateral flow assay on paper strips displays 100% agreement with fluorescence-based reporter assay in the clinical validation. Following a 30-minute pre-amplification RT-LAMP step, the lyophilized ENHANCEv2 is shown to achieve high sensitivity and specificity while reducing CRISPR reaction time to as low as 3 minutes and maintaining its detection capability upon storage at room temperature for several weeks.

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