Rapid detection of African swine fever virus based on lateral flow nucleic acid assay

Abstract: African swine fever virus (ASFV) was a devastating contagion of swine that could cause 100% mortality of infected animals. The outbreak of ASFV in China in 2019 had caused great economic losses in the pig industry. Therefore, simple and rapid diagnostic technology was highly needed for ASFV prevention. However, most of the current detection methods, such as protein-based technology and fluorescent quantitative polymerase chain reaction (PCR) technology, required complicated and expensive instruments, as well as professional technicians. Isothermal amplification technology did not require complex instruments but needed expensive reagents, which also limited its widespread usage, especially for laboratories with limited resources. In order to meet the needs of laboratories with limited resources to detect ASFV, a new low-cost lateral flow nucleic acid assay (LFNAA) was developed by combining the traditional PCR technique and the colloid gold lateral flow technique. LFNAA could observe the test results with naked eyes. A unique tailed-primer was designed to avoid the need for complicated antigen-labeling and expensive antibody, thus the PCR could produce a double-stranded DNA product with a single-strand oligonucleotide tail at one end, then the PCR product could hybridize with a colloidal gold-labeled oligonucleotide capture probe. In the presence of the positive product, the biotin on the other end of the product would bind to the streptavidin pre-immobilized on the test line of the strip and formed a sandwich structure of "streptavidin - biotin - PCR product - oligonucleotide tail - gold-labeled capture probe", so the test line of the strip would show a red color to indicate the positive result. On the other hand, if there was no positive product in the sample, the test line would show no red color. So, the detection result could be judged by observing whether the test line has a red color. Besides, the excess gold-labeled capture probe would hybridize with the control probe on the control line, showing a red color as an assay valid control. As a verification of the assay specificity, the LFNAA system was used to identify the presence of ASFV in actual samples of CSFV, PRRSV, PCV1 and PCV2, PRV, PPV. The result showed that the LFNAA could specifically detect ASFV, which was consistent with the fluorescent quantitative PCR. And the sensitivity of the LFNAA was the same with the agarose gel electrophoresis, both could reach to 103 copies/μL. Therefore, the LFNAA can meet the requirements of rapid and sensitive ASFV identification with only a common PCR instrument. Its rapidity (<2 h), low-cost, and simple-operation are greatly suitable for the non-professionals in the laboratories with limited resources. LFNAA avoids the usage of the expensive antibody and the complicated process of AuNP-antibody conjugation. Furthermore, due to the generality of the technique, the more simple isothermal amplification technology is expected to replace the current PCR method to make it more convenient and simple, and realizes faster and easier field testing in food safety testing and medical diagnosis.