Abstract:
Largemouth bass virus (LMBV) is a major viral pathogen in largemouth bass culture, usually causing high mortality and heavy economic losses. Accurate and early detection of LMBV is crucial for diagnosis and control of the diseases caused by LMBV. Previously, we selected the specific aptamers, LA38 and LA13, targeting LMBV by systematic evolution of ligands by exponential enrichment (SELEX). In this study, we further generated truncated LA38 and LA13 (named as LA38s and LA13s) with high specificity and affinities and developed an aptamer-based sandwich enzyme-linked apta-sorbent assay (ELASA) for LMBV diagnosis. The sandwich ELASA showed high specificity and sensitivity for the LMBV detection, without cross reaction with other viruses. The detection limit of the ELASA was as low as 1.25 × 102 LMBV-infected cells, and the incubation time of the lysate and biotin labeled aptamer was as short as 10 min. The ELASA could still detect LMBV infection in spleen lysates at dilutions of 1/25, with good consistency of qRT-PCR. For the fish samples collected from the field, the sensitivity of ELASA was 13.3% less than PCR, but the ELASA was much more convenient and less time consuming. The procedure of ELASA mainly requires washing and incubation, with completion in approximately 4 h. The sandwich ELASA offers a useful tool to rapidly detect LMBV rapidly, contributing to control and prevention of LMBV infection.
摘要:
大口鲈鱼病毒(LMBV)是大口鲈鱼养殖中的主要病毒病原体,通常造成高死亡率和沉重的经济损失。LMBV的准确和早期检测对于诊断和控制LMBV引起的疾病至关重要。以前,我们选择了特定的适体,LA38和LA13,通过指数富集(SELEX)配体的系统进化靶向LMBV。在这项研究中,我们进一步产生了具有高特异性和亲和力的截短LA38和LA13(命名为LA38s和LA13s),并开发了一种基于适体的夹心酶联apta吸附测定(ELASA)用于LMBV诊断.夹心ELASA对LMBV检测具有较高的特异性和敏感性,与其他病毒无交叉反应。ELASA的检测限低至1.25×102LMBV感染细胞,裂解物和生物素标记的适体的孵育时间短至10分钟。在1/25稀释度的脾脏裂解物中,ELASA仍然可以检测到LMBV感染,qRT-PCR具有良好的一致性。从田间采集的鱼样本,ELASA的灵敏度比PCR低13.3%,但是ELASA更方便,耗时更少。ELASA的程序主要需要洗涤和孵育,在大约4小时内完成。三明治ELASA提供了一个有用的工具来快速检测LMBV,有助于控制和预防LMBV感染。