利用侧流核酸试纸条快速检测非洲猪瘟病毒

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

    • 摘要: 为满足基层普通实验室或养殖场等资源有限的实验室对非洲猪瘟病毒(African swine fever virus, ASFV)的检测需求,该研究开发了一种简单、快速、低成本的检测技术,可以用肉眼观察检测结果。研究将传统聚合酶链式反应(polymerase chain reaction, PCR)与胶体金试纸条技术结合,开发一种低成本的侧流核酸测定试纸条(lateral flow nucleic acid assay, LFNAA)。该体系设计了独特的尾引物,避免了传统核酸试纸条的半抗原标记及抗体的使用。经过PCR扩增后产生一端带着单链寡核苷酸尾巴的双链DNA产物,能够与胶体金标记的寡核苷酸捕获探针结合,从而在试纸条上形成可以用肉眼观察的目标产物。该LFNAA试纸条能够在猪瘟病毒(Classical swine fever virus, CFSV)、猪瘟病毒猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)、猪圆环病毒1型(Porcine circovirus 1, PCV1)、猪圆环病毒2型(Porcine circovirus 2, PCV2)、猪伪狂犬病毒(Pseudorabies virus, PRV)、猪细小病毒(Porcine parvovirus virus, PPV)中特异的鉴定出ASFV的存在,其灵敏度与琼脂糖凝胶电泳的分析结果一致,均能达到103 copies/μL。因此,仅需要一台普通PCR仪,即可对ASFV进行快速灵敏的鉴定(<2 h),其低成本、操作简便的特点非常适合资源有限的实验室中非专业人员的操作。此外,该技术可进一步结合等温扩增技术,能够在食品安全和医学诊断中实现更快更简便的现场检测。

       

      Abstract: 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.

       

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