七种猪常见病毒核酸磁-纳米微粒可视化快速检测技术的建立与应用

doi:10.11843/j.issn.0366-6964.2023.09.024

摘要/Abstract

摘要： 随着集约化养殖业的发展,规模化养猪场对于病原的早期快速检测需求逐步增高,本研究旨在建立针对猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、猪瘟病毒(classical swine fever virus,CSFV)、猪圆环病毒2型(porcine circovirus type 2,PCV2)、猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)、猪伪狂犬病病毒(porcine pseudorabies virus,PRV)、猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)和猪细小病病毒(porcine parvovirus,PPV)等7种常见病毒病病原感染的广谱磁纳米微粒可视化(ultrasensitive nanoparticle visualization detection,UNVs)现场检测技术。根据不同病毒的复制酶区域的高度保守区域,设计探针和引物,通过探针的筛选和条件的优化,得到具有特异性强、富集效率和杂交效率高的目的病原基因序列特异性识别探针(富集探针和杂交探针),分别与四氧化三铁磁性微粒和纳米金偶联,制备成针对每种病原的特异性功能化磁珠和功能化纳米金。以功能化磁珠为载体富集核酸信号,功能化纳米金做桥梁放大信号,使用双探针同时检测病原核酸,结合碱性磷酸酶的酶促可视化反应检测信号,建立针对猪PCV2、PRV、PPV、PEDV、TGEV、PRRSV和CSFV等7种病毒的现场可视化UNVS便捷诊断技术。结果显示,筛选设计的核酸探针与猪的其它病原无交叉反应,特异性良好。建立的针对PCV2、PRV、PPV、PEDV、TGEV、PRRSV和CSFV病原的UNVs可视化检测技术,检测限为10³ copies·mL^-1(血清样本)或10³ copies·g^-1(粪便样本),基本上达到了PCR或RT-PCR的检测敏感度,灵敏度组内与组间最大变异系数均小于5%,显色结果稳定,无较大差异,有良好的重复性,单个样本的检测可在4 h以内完成。应用该技术和PCR检测对来自陕西省的499份临床样品(疑患有PCV2、PRV、PPV、PEDV、TGEV、PRRSV和CSFV的猪的血清、粪便样品)进行同步检测和复检,结果显示,建立的针对7种病原的UNVs可视化检测方法不仅具有与普通PCR相同的敏感度,甚至与普通PCR相比有更高的检出率。本研究成功建立了针对PCV2、PRV、PPV、PEDV、TGEV、PRRSV和CSFV病原的UNVs可视化检测技术,具有灵敏度较高、检测范围广以及特异性和可靠性良好的优点,为猪的多种病原提供了一种快速、准确、灵敏的诊断技术。

关键词: 猪病毒病, 磁纳米微粒, 碱性磷酸酶, 可视化, 现场检测

Abstract: With the development of intensive farming, the demand for early and rapid detection of various pathogens in large-scale pig farms has gradually increased. The aim of this study was to establish the broad-spectrum magnetic nanoparticle visualization detection (UNVs) field detection technology for porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), porcine epidemic diarrhea virus (PEDV), porcine pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), and porcine parvovirus (PPV). Based on the highly conserved regions of different viruses, probes and primers were designed, and sequence-specific identification probes (enrichment probes and hybridization probes) with high specificity and enrichment and hybridization efficiency for the target pathogen genes were obtained through the screening of probes and optimization of conditions, which were coupled with ferric oxide magnetic particles and nanogold, respectively, to prepare functionalized magnetic beads specific for each pathogen and functionalized nanogold. The functionalized magnetic beads were used as carriers to enrich nucleic acid signals, and functionalized nanogold was used as a bridge to amplify signals, and the dual probes were used to simultaneously detect pathogenic nucleic acids, combined with the enzymatic visualization reaction of alkaline phosphatase to detect signals, to establish a convenient diagnostic technique for on-site visualization of UNVS for seven kinds of viruses, including PCV2, PRV, PPV, PEDV, TGEV, PRRSV, and CSFV. The results showed that the nucleic acid probes designed for screening did not cross-react with other pathogens of pigs and had good specificity. The established technique for the visualization of UNVs for PCV2, PRV, PPV, PEDV, TGEV, PRRSV and CSFV pathogens has a detection limit of 10³copies·mL^-1 serum sample or 10³ copies·g^-1 stool sample, which basically achieves the sensitivity of PCR or RT-PCR detection, the maximum coefficient of variation within and between groups is less than 5%, the colorimetric results are stable without large differences, there is good reproducibility, and the detection of a single sample can be completed in less than 4 h. The results of simultaneous testing and retesting of 499 clinical samples (serum and fecal samples from pigs suspected of having PCV2, PRV, PPV, PEDV, TGEV, PRRSV and CSFV) from Shaanxi Province by applying the technique and PCR assay showed that the established visualization method for UNVs against seven pathogens not only had the same sensitivity as ordinary PCR, but even had a higher detection rate compared with ordinary PCR. In this study, we successfully established a visual detection technique for UNVs against PCV2, PRV, PPV, PEDV, TGEV, PRRSV and CSFV pathogens, which has the advantages of high sensitivity, wide detection range as well as good specificity and reliability, providing a rapid, accurate and sensitive diagnostic technique for a variety of pathogens in pigs.

Key words: porcine virus disease, magnetic nanoparticle, alkaline phosphatase, visualization, field test

中图分类号:

S858.285.3

赵永攀, 郑芳芳, 尹俊卿, 杜谦, 童德文, 黄勇. 七种猪常见病毒核酸磁-纳米微粒可视化快速检测技术的建立与应用[J]. 畜牧兽医学报, 2023, 54(9): 3848-3862.

ZHAO Yongpan, ZHENG Fangfang, YIN Junqing, DU Qian, TONG Dewen, HUANG Yong. Establishment and Application of Magnetic Nanoparticle Visualization Technique for Nucleic Acid Detection of Seven Common Swine Viruses[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3848-3862.

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