scholarly journals A CRISPR/Cas12a Based Universal Lateral Flow Biosensor for the Sensitive and Specific Detection of African Swine-Fever Viruses in Whole Blood

Biosensors ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 203
Author(s):  
Jinghua Wu ◽  
Omar Mukama ◽  
Wei Wu ◽  
Zhiyuan Li ◽  
Jean De Dieu Habimana ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
African Swine Fever ◽  
Food Samples ◽  
Lateral Flow ◽  
Economic Threat ◽  
Polymerase Chain ◽  
High Fluorescence ◽  
Safety Applications

Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful; however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR–Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 × 10−15 M within 2 h, and unambiguously identified ASFV from swine blood. This system uses less time for PCR pre-amplification and requires cheaper devices; thus, it can be applied to virus monitoring and food samples detection.

Combination of Immunomagnetic Separation and Polymerase Chain Reaction for the Simultaneous Detection of Listeria monocytogenes and Salmonella spp. in Food Samples

2001 ◽  
Vol 64 (11) ◽  
pp. 1744-1750 ◽  
Author(s):  
HSIEN-YEE HSIH ◽  
HAU-YANG TSEN
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Simultaneous Detection ◽  
Meat Products ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Pcr Method ◽  
Polymerase Chain

A method that combined the immunomagnetic separation (IMS) technique and the multiplex polymerase chain reaction (PCR) method (i.e., the IMS-mPCR method) was developed for simultaneous detection of Listeria monocytogenes and Salmonella spp. in food samples. When only the multiplex PCR method was used, it was found that if cell numbers of each of the two target organisms (L. monocytogenes and Salmonella spp.) were above the detection limit, but differed by more than 2 logs—e.g., n × 107 to n × 104 or n × 106 to n × 103—the organism presenting the lower numbers might go undetected. Following the enrichment step with universal preenrichment (UP) broth, if an IMS method using equal quantities of anti-Listeria and anti-Salmonella immunomagnetic beads was performed prior to PCR, both pathogens could be detected unambiguously. Such results could be obtained for target organisms in food samples, such as milk, dairy, and meat products, if similar enrichment and IMS steps were performed prior to PCR.

scholarly journals Arthropods as potential vectors of African swine fever virus outbreaks on pig farms in the Republic of Korea

2020 ◽  
Author(s):  
Hachung Yoon ◽  
Seong-Keun Hong ◽  
Ilseob Lee ◽  
Eune-Seob Lee
Keyword(s):  
Genomic Dna ◽  
Laboratory Tests ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Chain Reaction ◽  
Potential Vector ◽  
Pig Farms ◽  
Polymerase Chain ◽  
The Republic

The seasonality of African swine fever (ASF), with cases concentrated over the summer in Europe, in addition to outbreaks on farms with high levels of biosecurity, suggest that ASF virus (ASFV) may be transmitted by arthropod vectors. In this study, arthropods were collected from Korean pig farms with ASF outbreaks to determine the role of arthropods as a potential vector of ASFV. Arthropods were collected from 14 farms with ASF outbreaks, from September 27 to October 31, 2019. A total of 28,729 arthropods, including 28,508 (99.2%) Diptera, were collected using blacklight traps, insect nets, and yellow sticky strips. All arthropods samples were negative for ASFV genomic DNA according to laboratory tests using real-time polymerase chain reaction. Nevertheless, it is premature to conclude that arthropods do not play any role in ASFV transmission.

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 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.

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