A New Method for Detection African Swine Fever Virus: Time-resolved Fluorescence Immunoassay

Abstract

African swine fever (ASF) has severely influenced the swine industry of the whole world. Fast and accurate African swine fever virus (ASFV) antigen detection is very important for ASF prevention. This study aims to establish a new detection method for detection ASFV antigen using time-resolved fluorescence immunoassay (TRFIA) in the nose and mouth discharge. A double antibody sandwich TRFIA method was optimized and established. Recombinant P30 recombinant antigen was captured by its antibodies immobilized on 96-well plate, and then banded together with another detection antibodies labeled with Europium(III) (Eu 3+ ) chelates, finally time-resolved analyzer measured the fluorescence intensity. The performance of this TRFIA (sensitivity, specificity and accuracy) was evaluated using the clinical samples and compared with the nucleic acid testing method. The sensitivity of this TRFIA was 0.015 ng/mL (dynamic range 0.24–500 ng/mL) with high specificity. The recovery ranged from 92.00 to 103.62 %, the inter-assay CVs ranged from 5.50 to 11.96 %, and the intra-assay CVs was between 5.20 and 10.53 %. Additionally, the cutoff value was 0.016. TRFIA took only 45 min to generate results, and its detection capability comparable to the nucleic acid detection. This study developed a TRFIA method that could be used for qualitative/quantitative detection of ASFV antigen in pigs nasal discharge, which has high sensitivity, specificity and accuracy. This TRFIA provides a new method for rapidly screening ASFV infection in pigs industry.

A New Method for Early Screening of Gastric Cancer (G17 and CA724 Dual-Labeled Time-Resolved Fluorescence Immunoassay)

“…Early/primary screening of the patients with atrophic gastritis and early gastric cancer by endoscopy is unrealistic, and noninvasive serological screening is a practical method and should be further validated [14]. Time-resolved fluorescence immunoassay (TRFIA) is a novel detection technique with high sensitivity, lower matrix interference, and enlarge dynamic range, and it has been used in the diagnosis and screening of various human diseases [15][16][17]. However, TRFIA has not yet been used in G-17 and CA724 detection.…”

The Development of a Real-Time Recombinase-Aid Amplification Assay for Rapid Detection of African Swine Fever Virus

“…The sensitivity of the time-resolved fluorescence immunoassay (TRFIA) for ASFV antigen detection was 0.015 ng/ml. The protocol time was only 45 min, which is suitable for the laboratory diagnosis of ASFV infection ( Chen et al, 2021 ). Together, the above-mentioned methods are time-consuming and unsuitable for rapid detection on-site.…”

Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for detection of African swine fever virus (ASFV)

“…2 ). The pUC57-p72 DNA was used as the template for optimization of the LMTIA system and determination of the sensitivity, as a standard ASFV plasmid was used in previous reports [ 5 18 ]. Genomic DNAs (gDNAs) extracted from 200 swine serum samples were provided by Henan Tuodao Biotechnology Co., Ltd., China, and these DNAs were used for comparison of the LMTIA assay and the commercial real-time PCR kit (Zhengzhou Zhongdao Biotechnology Co., Ltd., China).…”

“…Many modern molecular biology techniques have been used for ASFV detection. The assays based on immunology include colloidal gold test strip assay [ 4 ], time-resolved fluorescence immunoassay [ 5 ], fluorescent immunochromatography test strip [ 6 ], amplified luminescent proximity homogeneous assay (AlphaLISA) [ 7 ], triplex bead-based assay for the simultaneous detection of antibodies [ 8 ], Eu (III) chelate microparticle-based lateral flow test strip and blocking enzyme-linked immunosorbent assay [ 2 9 ]. Many nucleic acid amplification methods have been improved or modified for the detection of ASFV, such as multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay [ 10 ], triplex real-time PCR assay [ 11 ], real-time loop-mediated isothermal amplification (LAMP) assay [ 12 13 ], LAMP combined with lateral flow dipstick [ 14 ], semi-quantitative colorimetric LAMP [ 15 ], recombinase polymerase amplification (RPA) combined with lateral flow [ 16 17 18 ], PCR with a lateral flow strip [ 19 ], improved real-time PCR system with probe modification [ 20 ], CRISPR-Cas12a system coupled with PCR or LAMP [ 21 ], recombinase aided amplification combined with CRISPR/Cas12a [ 22 ], RPA-CRISPR-based nucleic acid detection method [ 23 ] and Crispr/cas12a technology combined with immunochromatographic strips [ 24 ].…”