scholarly journals Cas12a-based on-site and rapid nucleic acid detection of African swine fever

2019 ◽  
Author(s):  
Jing Bai ◽  
Haosi Lin ◽  
Haojian Li ◽  
Yang Zhou ◽  
Junshan Liu ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lateral Flow ◽  
Ease Of Use ◽  
Nucleic Acid Detection ◽  
Lateral Flow Strip ◽  
Trade Offs ◽  
Laboratory Facilities ◽  
Sensitivity Specificity

AbstractThe mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and is caused by African swine fever virus (ASFV), can reach 100%. ASF has been reported in 25 Chinese provinces since August 2018. There is no effective treatment or vaccine for it and the present molecular diagnosis technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-Cas12a-based system that combines recombinase-aided amplification (RAA) and CRISPR/Cas12a for ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in the RAA-Cas12a based system (named CORDS, Cas12a-based On-site and Rapid Detection System). We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout. CORDS could identify the p72 gene at femtomolar sensitivity in an hour at 37°C, and only requires an incubator. For ease of use, the regents of CORDS was lyophilized to three tubes and remained the same sensitivity when stored at 4 °C for at least 7 days. Thus, CORDS provides a rapid, sensitive and easily operable method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and storage, and is ready for field applications.

scholarly journals LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

2021 ◽  
Author(s):  
Bo YANG ◽  
zhengwang shi ◽  
Yuan Ma ◽  
Lijuan Wang ◽  
Liyan Cao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection System ◽  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Portable Detection ◽  
Real Time Qpcr

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporters and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. In the current study, a LAMP coupled with the CRISPR detection method was developed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

scholarly journals An Isothermal Molecular Point of Care Testing for African Swine Fever Virus Using Recombinase-Aided Amplification and Lateral Flow Assay Without the Need to Extract Nucleic Acids in Blood

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuhang Zhang ◽  
Qingmei Li ◽  
Junqing Guo ◽  
Dongliang Li ◽  
Li Wang ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
Lateral Flow ◽  
Fever Virus ◽  
Lateral Flow Assay ◽  
Economic Losses ◽  
Point Of Care Testing ◽  
Viral Dna ◽  
Blood Samples

African swine fever (ASF) is a highly contagious and usually deadly porcine infectious disease listed as a notifiable disease by the World Organization for Animal Health (OIE). It has brought huge economic losses worldwide, especially since 2018, the first outbreak in China. As there are still no effective vaccines available to date, diagnosis of ASF is essential for its surveillance and control, especially in areas far from city with limited resources and poor settings. In this study, a sensitive, specific, rapid, and simple molecular point of care testing for African swine fever virus (ASFV) B646L gene in blood samples was established, including treatment of blood samples with simple dilution and boiling for 5 min, isothermal amplification with recombinase-aided amplification (RAA) at 37°C in a water bath for 10 min, and visual readout with lateral flow assay (LFA) at room temperature for 10–15 min. Without the need to extract viral DNA in blood samples, the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min. The detection limit of RAA-LFA for synthesized B646L gene-containing plasmid was 10 copies/μl, which was 10-fold more sensitive than OIE-recommended PCR and quantitative PCR. In addition, no positive readout of RAA-LFA was observed in testing classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, pseudorabies virus and porcine circovirus 2, exhibiting good specificity. Evaluation of clinical blood samples of RAA-LFA showed 100% coincident rate with OIE-recommended PCR, in testing both extracted DNAs and treated bloods. We also found that some components in blood samples greatly inhibited PCR performance, but had little effect on RAA. Inhibitory effect can be eliminated when blood was diluted at least 32–64-fold for direct PCR, while only a 2–4 fold dilution of blood was suitable for direct RAA, indicating RAA is a better choice than PCR when blood is used as detecting sample. Taken together, we established an sensitive, specific, rapid, and simple RAA-LFA for ASFV molecular detection without the need to extract viral DNA, providing a good choice for point of care testing of ASF diagnosis in the future.

scholarly journals CASLFA: CRISPR/Cas9-mediated lateral flow nucleic acid assay

2019 ◽  
Author(s):  
Xusheng Wang ◽  
Erhu Xiong ◽  
Tian Tian ◽  
Meng Cheng ◽  
Wei Lin ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
Analytical Techniques ◽  
High Specificity ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Nucleic Acid Assay ◽  
Ancillary Equipment

AbstractThe lateral flow assay is one of the oldest and most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the tedious and inefficient hybridization steps. Here, we have introduced a new version of the lateral flow assay, termed Cas9-mediated lateral flow nucleic acids assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify Listeria monocytogenes, genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a sensitivity of hundreds of copies of genome samples with high specificity within 1 h. CASLFA satisfies some of the characteristics of a next-generation molecular diagnostics tool due to its rapidity and accuracy, allowing for point-of-care use without the need for technical expertise and complex ancillary equipment. This method has great potential for analyzing genes in resource-poor or nonlaboratory environments.

The Detection of Tuberculosis by Loop-Mediated Isothermal Amplification (LAMP) Combined with a Lateral Flow Dipstick

2015 ◽  
pp. 269-300
Author(s):  
Thongchai Kaewphinit ◽  
Somchai Santiwatanakul ◽  
Kosum Chansiri
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Lateral Flow ◽  
Ease Of Use ◽  
Isothermal Amplification ◽  
Toxic Substances ◽  
Nested Polymerase Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Lateral Flow Dipstick ◽  
Electrophoresis Method ◽  
Sensitivity Specificity

Tuberculosis (TB) is an airborne infectious disease caused by the bacterium Mycobacterium Tuberculosis (MTB) and is a persistent problem in developing countries. Present methods for its detection include normal or nested Polymerase Chain Reaction (PCR) followed by electrophoresis, real-time PCR, Ziehl-Neelsen staining, and culture assay. These techniques entail various disadvantages such as high cost, long assay time and use of toxic substances. Novel loop-mediated isothermal amplification (LAMP) permits DNA to be amplified rapidly under constant temperature. The combination of LAMP and chromatographic Lateral Flow Dipstick (LAMP-LFD) by using biotinylated LAMP amplicon hybridized with Fluorescein Isothiocyanate (FITC)-labeled probes are allowed to detect MTB without electrophoresis and interpreted within 3-5 min. LAMP-LFD is as highly sensitive as PCR-electrophoresis method. Based on its sensitivity, specificity, rapidity, cost effectiveness, ease of use, and convenience, LAMP-LFD could be suitable for use in early MTB detection.

scholarly journals No part gets left behind: Tiled nanopore sequencing of whole ASFV genomes stitched together using Lilo

2021 ◽  
Author(s):  
Amanda Warr ◽  
Caitlin Newman ◽  
Nicky Craig ◽  
Ingrida Vendelė ◽  
Rizalee Pilare ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Low Cost ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
African Swine Fever ◽  
Pork Production ◽  
Long Time ◽  
Optimal Efficiency ◽  
Public Repositories ◽  
High Level

AbstractAfrican Swine Fever virus (ASFV) is the causative agent of a deadly, panzootic disease, infecting wild and domesticated suid populations. Contained for a long time to the African continent, an outbreak of a particularly infectious variant in Georgia in 2007 initiated the spread of the virus around the globe, severely impacting pork production and local economies. The virus is highly contagious and has a mortality of up to 100% in domestic pigs. It is critical to track the spread of the virus, detect variants associated with pathology, and implement biosecurity measures in the most effective way to limit its spread. Due to its size and other limitations, the 170-190kbp large DNA virus has not been well sequenced with fewer than 200 genome sequences available in public repositories. Here we present an efficient, low-cost method of sequencing ASFV at scale. The method uses tiled PCR amplification of the virus to achieve greater coverage, multiplexability and accuracy on a portable sequencer than achievable using shotgun sequencing. We also present Lilo, a pipeline for assembling tiled amplicon data from viral or microbial genomes without relying on polishing against a reference, allowing for structural variation and hypervariable region assembly other methods fail on. The resulting ASFV genomes are near complete, lacking only parts of the highly repetitive 3’- and 5’telomeric regions, and have a high level of accuracy. Our results will allow sequencing of ASFV at optimal efficiency and high throughput to monitor and act on the spread of the virus.

scholarly journals TECHNICAL CONSIDERATIONS IN LOW-COST HERITAGE DOCUMENTATION

2019 ◽  
Vol XLII-2/W17 ◽  
pp. 225-232
Author(s):  
A. Murtiyoso ◽  
P. Grussenmeyer ◽  
D. Suwardhi
Keyword(s):  
Point Cloud ◽  
Low Cost ◽  
Bundle Adjustment ◽  
Ease Of Use ◽  
Cloud Data ◽  
Heritage Sites ◽  
Common Reference ◽  
Trade Offs ◽  
Dense Point

Abstract. The use of photogrammetry in 3D heritage documentation has matured over the recent years. In the same time, many types of sensors have also been developed in the field of imaging. While photogrammetry is considered as a low-cost alternative to TLS, several options exist in terms of sensor type with trade-offs between price, ease of use, and quality of resolution. Nevertheless, a proper knowledge on the acquisition and processing is still required to generate acceptable results. This paper aims to compare three photogrammetric sensors, namely a classical DSLR camera, a drone, and a spherical 360° camera in documenting heritage sites. Main comparison points include quality of the bundle adjustment and quality of the dense point cloud. However, an important point of the paper is also to determine whether a sensor at a given cost and effort is enough for documentation purposes. A TLS point cloud data was used as a common reference, as well as control and check points issued from geodetic surveying. In the aftermath of the comparison, several technical suggestions and recommendations were proposed as regards to the use of each sensor.

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