Lethbridge Laboratory, National Centres for Animal Disease, Canadian Food Inspection Agency, Lethbridge, AB, Canada.
Nexogen, Inc., San Diego, CA, USA.
Transbound Emerg Dis. 2018 Apr;65(2):e272-e283. doi: 10.1111/tbed.12749. Epub 2017 Nov 30.
Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases.
微阵列技术在病原体检测方面很有用，因为它可以同时检测大量遗传特征的存在或缺失。然而，大多数微阵列检测方法在执行过程中既费力又耗时。本研究描述了一种多重逆转录（RT）-PCR 和新型配套自动化电子微阵列检测方法的开发和初步评估，用于同时检测和区分七种影响猪的重要病毒，包括口蹄疫病毒（FMDV）、猪水疱病病毒（SVDV）、猪水疱疹病毒（VESV）、非洲猪瘟病毒（ASFV）、经典猪瘟病毒（CSFV）、猪呼吸道和繁殖综合征病毒（PRRSV）和猪圆环病毒 2 型（PCV2）。新型电子微阵列检测方法使用单个用户友好的仪器，该仪器集成并自动化了在一次性电子微阵列盒上进行捕获探针打印、杂交、洗涤和报告的功能，该微阵列盒具有 400 个特征。该检测方法准确地检测和鉴定了总共 68 株七种目标病毒株，包括 23 株口蹄疫病毒，代表所有七种血清型，以及 10 株 CSFV 株，代表所有三种基因型。该检测方法成功地从田间临床样本、实验感染动物（FMDV 和 SVDV 最早为感染后 1 天，ASFV 为 4 天，CSFV 为 5 天）以及用目标病毒接种的生物材料中检测到病毒。ASFV、PCV2 和 PRRSV 的检测限为 10 拷贝/μl，SVDV、CSFV、VESV 的检测限为 100 拷贝/μl，FMDV 的检测限为 1,000 拷贝/μl。与多重 RT-PCR 相比，电子微阵列组件对几种目标病毒的分析灵敏度降低。捕获探针打印的集成允许根据需要进行现场定制的阵列打印，而电泳驱动的杂交比依赖被动杂交的传统微阵列更快地产生结果。经过进一步改进，这种新型快速高度自动化的微阵列技术具有在牲畜疾病多病原体监测方面的潜在应用。
