Validation efficiency of coupling of beads and probes, used in high-throughput multiplexed nucleic acid detection of respiratory and reproductive pathogens in swine

This file set contains results files from the Bio-Plex integrated biomolecular assay system, each containing the data from a reading, the Protocol parameters used to collect that data and analysis tools for interpreting the data. The .rbx format data files must be opened by the Bio-Plex Manager integrated software. Please see the references link below for .xls format data related to this study.<br><br>Magnetic carboxylic beads were coupled to different virus probes as follows: PRV (no. 26), PRRSV (no. 29), CSFV (no. 34), JEV (no. 36), PCV-2 (no. 46), ASFV (no. 62) and PPV (no. 65). <br>Validation of coupling was performed with different amounts of biotin-modified RCS. The curve was drawn with 0, 5, 10, 20, 50, 100 and 200 fmol RCS, respectively.<br>Article abstract:<br>Background: The aim of this study was to develop a multiple PCR assay based on the suspension array system for the simultaneous detection of respiratory and reproductive pathogens in swine. Pseudorabies virus (PRV), Japanese encephalitis virus (JEV), classic swine fever virus (CFSV), African swine fever virus (ASFV), porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) are the major respiratory and reproductive viral pathogens in pig farms.<br>Results: Seven pairs of specific primers and probes were designed, and multiple PCR was performed, with the PCR products hybridized to beads coupled to probes, which were then detected by Bio-Plex suspension array system. The limit of detection, specificity and repeatability of this method was determined. The assay was further tested using 137 clinical samples, and the results were compared with conventional PCR to evaluate the ability of the method to diagnose porcine viruses. The results showed that the assay had a high degree of specificity and repeatability, and the simultaneous detection limit for the seven viruses reached 103 copies/μL. The detection of viruses in clinical samples using the Bio-Plex method was high and comparable to conventional PCR (&gt; 90%).

本文件集包含Bio-Plex集成生物分子检测系统的结果文件，每份文件均包含单次检测所得数据、采集该数据时所用的实验方案参数，以及用于解读该数据的分析工具。格式为.rbx的数据文件需通过Bio-Plex Manager集成软件打开。如需获取本研究相关的.xls格式数据，请参阅下方参考文献链接。

将磁性羧基磁珠与不同病毒探针偶联，具体如下：伪狂犬病病毒（Pseudorabies virus, PRV，编号26）、猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV，编号29）、猪瘟病毒（Classical swine fever virus, CSFV，编号34）、日本脑炎病毒（Japanese encephalitis virus, JEV，编号36）、猪圆环病毒2型（Porcine circovirus type 2, PCV-2，编号46）、非洲猪瘟病毒（African swine fever virus, ASFV，编号62）及猪细小病毒（Porcine parvovirus, PPV，编号65）。

采用不同剂量的生物素修饰RCS对磁珠偶联效果进行验证，并分别以0、5、10、20、50、100及200 fmol的RCS绘制标准曲线。

论文摘要：
背景：本研究旨在开发一种基于悬浮阵列系统的多重PCR检测方法，用于同时检测猪呼吸道与生殖道病原体。伪狂犬病病毒（PRV）、日本脑炎病毒（JEV）、猪瘟病毒（CFSV）、非洲猪瘟病毒（ASFV）、猪圆环病毒2型（PCV-2）、猪繁殖与呼吸综合征病毒（PRRSV）及猪细小病毒（PPV）为猪场中主要的呼吸道与生殖道病毒性病原体。
结果：本研究设计了7对特异性引物与探针，开展多重PCR扩增，将PCR产物与偶联了探针的磁珠进行杂交，随后通过Bio-Plex悬浮阵列系统完成检测。对该方法的检测限、特异性与重复性进行了测定。进一步使用137份临床样本对该检测方法进行验证，并与传统PCR方法对比，以评估其诊断猪病毒感染的性能。结果显示，该检测方法具有优异的特异性与重复性，对7种病毒的同时检测限可达10³ copies/μL。采用Bio-Plex方法检测临床样本中的病毒，其检出率较高，与传统PCR方法的一致性超过90%。