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.

scholarly journals Lateral Flow Assay for the Detection of African Swine Fever Virus Antibodies Using Gold Nanoparticle-Labeled Acid-Treated p72

2022 ◽  
Vol 9 ◽  
Author(s):  
Wenzhuang Zhu ◽  
Kaiwen Meng ◽  
Yueping Zhang ◽  
Zhigao Bu ◽  
Dongming Zhao ◽  
...  
Keyword(s):  
Gold Nanoparticle ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
High Sensitivity ◽  
Lateral Flow ◽  
Fever Virus ◽  
Lateral Flow Assay ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Antibody Detection

African swine fever is a widespread and highly contagious disease in the porcine population, which is caused by African swine fever virus (ASFV). The PCR and ELISA detection methods are the main conventional diagnostic methods for ASFV antigen/antibody detection in the field. However, these methods have limitations of expensive equipment, trained technicians, and time-consuming results. Thus, a rapid, inexpensive, accurate and on-site detection method is urgently needed. Here we describe a double-antigen-sandwich lateral-flow assay based on gold nanoparticle-conjugated ASFV major capsid protein p72, which can detect ASFV antibody in serum samples with high sensitivity and specificity in 10 min and the results can be determined by naked eyes. A lateral flow assay was established by using yeast-expressed and acid-treated ASFV p72 conjugated with gold nanoparticles, which are synthesized by seeding method. A high coincidence (97.8%) of the assay was determined using clinical serum compared to a commercial ELISA kit. In addition, our lateral flow strip can detect as far as 1:10,000 diluted clinically positive serum for demonstration of high sensitivity. In summary, the assay developed here was shown to be rapid, inexpensive, accurate and highly selective. It represents a reliable method for on-site ASFV antibody detection and may help to control the ASFV pandemic.

scholarly journals Computational Analysis of African Swine Fever Virus Protein Space for the Design of an Epitope-Based Vaccine Ensemble

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1078 ◽  
Author(s):  
Albert Ros-Lucas ◽  
Florencia Correa-Fiz ◽  
Laia Bosch-Camós ◽  
Fernando Rodriguez ◽  
Julio Alonso-Padilla
Keyword(s):  
T Cell ◽  
Vaccine Development ◽  
De Novo ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Virus Protein ◽  
Hemorrhagic Disease ◽  
Starting Point

African swine fever virus is the etiological agent of African swine fever, a transmissible severe hemorrhagic disease that affects pigs, causing massive economic losses. There is neither a treatment nor a vaccine available, and the only method to control its spread is by extensive culling of pigs. So far, classical vaccine development approaches have not yielded sufficiently good results in terms of concomitant safety and efficacy. Nowadays, thanks to advances in genomic and proteomic techniques, a reverse vaccinology strategy can be explored to design alternative vaccine formulations. In this study, ASFV protein sequences were analyzed using an in-house pipeline based on publicly available immunoinformatic tools to identify epitopes of interest for a prospective vaccine ensemble. These included experimentally validated sequences from the Immune Epitope Database, as well as de novo predicted sequences. Experimentally validated and predicted epitopes were prioritized following a series of criteria that included evolutionary conservation, presence in the virulent and currently circulating variant Georgia 2007/1, and lack of identity to either the pig proteome or putative proteins from pig gut microbiota. Following this strategy, 29 B-cell, 14 CD4+ T-cell and 6 CD8+ T-cell epitopes were selected, which represent a starting point to investigating the protective capacity of ASFV epitope-based vaccines.

Genetic variation of african swine fever virus: Variable regions near the ends of the viral DNA

Virology ◽  
1989 ◽  
Vol 173 (1) ◽  
pp. 251-257 ◽  
Author(s):  
Rafael Blasco ◽  
Inmaculada de la Vega ◽  
Fernando Almazan ◽  
Montserrat Aguero ◽  
Eladio Viñuela
Keyword(s):  
Genetic Variation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Viral Dna ◽  
Variable Regions

scholarly journals Lateral flow assays for the detection of African swine fever virus antigen are not fit for field diagnosis of wild boar carcasses

2021 ◽  
Author(s):  
Paul Deutschmann ◽  
Jutta Pikalo ◽  
Martin Beer ◽  
Sandra Blome
Keyword(s):  
Wild Boar ◽  
Point Of Care ◽  
African Swine Fever ◽  
Virus Antigen ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Field Conditions ◽  
Real Field ◽  
Data Set ◽  
Field Samples

African swine fever (ASF) is one of the most important viral diseases of domestic pigs and wild boar. Apart from endemic cycles in Africa, ASF is now continuously spreading in Europe and Asia. As ASF leads to severe but unspecific clinical signs and high lethality, early pathogen detection is of utmost importance. Recently, “point-of-care” (POC) tests have been intensively discussed for the use in remote areas but also in the context of on-farm epidemiological investigations and wild boar carcass screening. Along these lines, the INGEZIM ASFV CROM Ag lateral flow assay (Eurofins Technologies Ingenasa) promises virus antigen detection under field conditions within minutes. In the present study, we evaluated the performance of the assay with selected high-quality reference blood samples, and also with real field samples from wild boar carcasses in different stages of decay from the ongoing ASF outbreak in Germany. While we observed a sensitivity of roughly 77% in freeze-thawed matrices of close to ideal quality, our approach to simulate field conditions in direct carcass testing without any modification resulted in a drastically reduced sensitivity of only 12.5%. Freeze thawing increased the sensitivity to roughly 44% which mirrored the overall sensitivity of 49% in the total data set of carcass samples. A diagnostic specificity of 100% was observed. However, most of the German ASF cases in wild boar would have been missed using the lateral flow assay (LFA) alone. Therefore, the antigen-specific LFA should not be regarded as a substitute for any OIE listed diagnostic method and has very limited use for carcass testing at the point of care. For optimized LFA antigen tests, the sensitivity with field samples must be significantly increased. An improved sensitivity is seen with freeze-thawed samples, which may indicate problems in the accessibility of ASFV antigen.

A Self-Contained Chemiluminescent Lateral Flow Assay for Point-of-Care Testing

2018 ◽  
Vol 90 (15) ◽  
pp. 9132-9137 ◽  
Author(s):  
Jinqi Deng ◽  
Mingzhu Yang ◽  
Jing Wu ◽  
Wei Zhang ◽  
Xingyu Jiang
Keyword(s):  
Point Of Care ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Point Of Care Testing

scholarly journals Development of a Directly Visualized Recombinase Polymerase Amplification–SYBR Green I Method for the Rapid Detection of African Swine Fever Virus

2020 ◽  
Vol 11 ◽  
Author(s):  
Shuai Zhang ◽  
Aijun Sun ◽  
Bo Wan ◽  
Yongkun Du ◽  
Yanan Wu ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Sybr Green ◽  
Fever Virus ◽  
Diagnostic Methods ◽  
Sybr Green I ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Field Samples ◽  
Standard Plasmid

African swine fever (ASF) is a lethal disease in swine caused by etiologic African swine fever virus (ASFV). The global spread of ASFV has resulted in huge economic losses globally. In the absence of effective vaccines or drugs, pathogen surveillance has been the most important first-line intervention to prevent ASF outbreaks. Among numerous diagnostic methods, recombinase polymerase amplification (RPA)-based detection is capable of producing sensitive and specific results without relying on the use of expensive instruments. However, currently used gene-specific, probe-based RPA for ASFV detection is expensive and time-consuming. To improve the efficiency of ASFV surveillance, a novel directly visualized SYBR Green I-staining RPA (RPAS) method was developed to detect the ASFV genome. SYBR Green I was added to the amplified RPA products for direct visualization by the naked eye. The sensitivity and specificity of this method were confirmed using standard plasmid and inactivated field samples. This method was shown to be highly specific with a detection limit of 103 copies/μl of ASFV in 15 min at 35°C without any cross-reactions with other important porcine viruses selected. In summary, this method enables direct sample visualization with reproducible results for ASFV detection and hence has the potential to be used as a robust tool for ASF prevention and control.

scholarly journals A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1129 ◽  
Author(s):  
Ferenc Olasz ◽  
István Mészáros ◽  
Szilvia Marton ◽  
Győző L. Kaján ◽  
Vivien Tamás ◽  
...  
Keyword(s):  
Sample Preparation ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Fever Virus ◽  
Whole Genome ◽  
Swine Industry ◽  
Viral Dna ◽  
Simple Method ◽  
Generation Sequencing

In the recent years, African swine fever has become the biggest animal health threat to the swine industry. To facilitate quick genetic analysis of its causative agent, the African swine fever virus (ASFV), we developed a simple and efficient method for next generation sequencing of the viral DNA. Execution of the protocol does not demand complicated virus purification steps, enrichment of the virus by ultracentrifugation or of the viral DNA by ASFV-specific PCRs, and minimizes the use of Sanger sequencing. Efficient DNA-se treatment, monitoring of sample preparation by qPCR, and whole genome amplification are the key elements of the method. Through detailed description of sequencing of the first Hungarian ASFV isolate (ASFV_HU_2018), we specify the sensitive steps and supply key reference numbers to assist reproducibility and to facilitate the successful use of the method for other ASFV researchers.

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