Ultrasensitive Detection and Subtyping of Porcine Endogenous Retrovirus Provirus Based on Magnetic Nanoparticles and Chemiluminescence.

Authors:
  • HYHaowen Yang
    Haowen Yang
  • ZLZhiyang Li
    Zhiyang Li
  • QJQinyang Jiang
    Qinyang Jiang
  • JFJing Fan
    Jing Fan
  • BZBingcong Zhou
    Bingcong Zhou
  • YGYafen Guo
    Yafen Guo
  • GLGanqiu Lan
    Ganqiu Lan
  • XYXiurong Yang
    Xiurong Yang
  • NHNongyue He
    Nongyue He
  • HJHesheng Jiang
    Hesheng Jiang
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Abstract
Porcine endogenous retrovirus (PERV) is commonly integrated in pig genomes, and could cause a cross-species infection by xenotransplantation. In this study, we developed a rapid and ultrasensitive approach for detection and subtyping of PERV provirus based on magnetic nanoparticles (MNPs) and chemiluminescence (CL). The carboxylated MNPs (CMNPs) were covalently coupled with aminated probes for capturing biotinylated target fragments of PERV, the product of polymerase chain reaction (PCR). Agarose gel electrophoresis analysis approved the reliability of biotinylated fragments. The MNPs composites were incubated with streptavidin-alkaline phosphatase (SA-ALP) and CL signal intensities were determined by subsequently adding 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD). The optimal assay conditions of this approach were 1 mM for SA modification, 10 μM for probe modification, 55 (PERV), 54 (PERV-A), 50 (PERV-B), and 56 °C (PERV-C) for hybridization temperatures respectively, and 30 min for hybridization time. This approach was specific and highly sensitive, and the limit of detection (LOD) was 100 amol, which has the potential for screening out safe pig donors for xenotransplantation as well as to examine clinical samples from human patients treated with porcine xenotranplantation.