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.

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 A miniPCR-duplex Lateral Flow Dipstick Platform in Combination with the Microfluidic Device for Rapid and Visual Diagnosis of Lymphatic Filariae Infection

2021 ◽  
Author(s):  
Achinya Phuakrod ◽  
Witsaroot Sripumk ◽  
Wutthinan Jeamsaksiri ◽  
Pattaraluck Pattamang ◽  
Sumart Loymek ◽  
...  
Keyword(s):  
Lymphatic Filariasis ◽  
Species Identification ◽  
Microfluidic Device ◽  
Lateral Flow ◽  
Detection Accuracy ◽  
Diagnostic Assay ◽  
Sequence Alignments ◽  
Blood Samples ◽  
Lateral Flow Dipstick ◽  
Visual Diagnosis

Abstract Background: Lymphatic filariasis is a major neglected tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. Accordingly, access is now needed to a cost-efficient, non-laborious diagnostic assay(s) capable of detecting low levels of microfilariae and without the need for infrastructure and expensive equipment.Methods: We developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD) assay. The design of the PCR primers was guided by sequence alignments of the HhaI and SspI genes of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection, accuracy, and cross-reactivity of the assay were evaluated, after which the assay was deployed to assess infection using blood samples obtained from Myanmar study participants living along the Thailand-Myanmar border, a region in Tak province endemic W. bancrofti. In addition, blood samples were provided by from Thais in Narathiwat province residing in an area endemic for brugian filariasis. We also combined a previously reported semi-automated microfluidic device with the miniPCR-DLFD to facilitate rapid detection and species identification of microfilariae in human blood.Results: The miniPCR-DLFD assay exhibited a detection limit of two microfilariae per mililiter blood sample, and cross-amplification was not observed with from other parasites. For field validation, microfilariae and DNA of W. bancrofti were detected from two (0.6%) and five (1.5%) out of 328 (100%) blood samples, respectively. For the Narathiwat samples, microfilariae and DNA of B. malayi were detected from one (0.46%) and two (0.9%) out of 216 (100%) blood samples, respectively. A rapid and visual of species identification was accurately obtained in all microfilariae entrapped by our previously developed microfluidic device.Conclusion: A miniPCR-DLFD platform alone or coupled with the microfluidic device provided rapid and visual diagnosis of lymphatic filariasis. The microfluidic device, the miniPCR, and the DLFD are all portable. Coupled with a portable microcentrifuge for DNA extraction, this is a promising platform for the diagnosis of lymphatic filariasis in point-of-collection settings with a modest cost (~USD $ 5) per sample.

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.

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