A strip of lateral flow gene assay using gold nanoparticles for point-of-care diagnosis of African swine fever virus in limited environment

2021 ◽  
Author(s):  
Zhiying Wang ◽  
Wenjie Yu ◽  
Ruibin Xie ◽  
Shuming Yang ◽  
Ailiang Chen
Keyword(s):  
Gold Nanoparticles ◽  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
Lateral Flow ◽  
Fever Virus ◽  
Gene Assay

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.

Microfluidic-CFPA Chip for the Point-of-Care Detection of African Swine Fever Virus with a Median Time to Threshold in about 10 min

ACS Sensors ◽  
2019 ◽  
Vol 4 (11) ◽  
pp. 3066-3071 ◽  
Author(s):  
Xin Ye ◽  
Lin Li ◽  
Jian Li ◽  
Xiaodong Wu ◽  
Xueen Fang ◽  
...  
Keyword(s):  
Median Time ◽  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
Fever Virus ◽  
Point Of Care Detection

Rapid on-site detection of African swine fever virus using polymerase chain reaction with a lateral flow strip

2020 ◽  
Vol 156 ◽  
pp. 104940 ◽  
Author(s):  
Dexin Zeng ◽  
Bingxu Qian ◽  
Kaiying Zhao ◽  
Yingjuan Qian ◽  
Wei Chen ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Lateral Flow ◽  
Fever Virus ◽  
Chain Reaction ◽  
Lateral Flow Strip ◽  
Polymerase Chain

scholarly journals Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254815
Author(s):  
Jinyu Fu ◽  
Yueping Zhang ◽  
Guang Cai ◽  
Geng Meng ◽  
Shuobo Shi
Keyword(s):  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
High Sensitivity ◽  
Fluorescence Assay ◽  
Fever Virus ◽  
Diagnostic Methods ◽  
High Morbidity ◽  
Swine Industry ◽  
Control And Prevention

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.

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.

scholarly journals High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

2019 ◽  
Author(s):  
Qian He ◽  
Dongmei Yu ◽  
Mengdi Bao ◽  
Grant Korensky ◽  
Juhong Chen ◽  
...  
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
High Throughput ◽  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
Fever Virus ◽  
Solution Phase ◽  
Nucleic Acid Amplification ◽  
Dna Complex

AbstractHere we report the development of a high throughput, all-solution phase, and isothermal detection system to detect African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with fluorescence-based point-of-care (POC) system we developed for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 hrs. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 hrs of incubation, resulting in an improvement of the detection limit to 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact, multiplexing, and simple ASFV detection, intended for low resource settings.

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