Ultrasensitive Detection of Porcine Epidemic Diarrhea Virus from Fecal Samples Using Functionalized Nanoparticles

Abstract

Porcine epidemic diarrhea virus (PEDV) is the main causative agent of porcine diarrhea, which has resulted in devastating damage to swine industry and become a perplexed global problem. PEDV infection causes lesions and clinical symptoms, and infected pigs often succumb to severe dehydration. If there is not a timely and effective method to control its infection, PEDV will spread rapidly across the whole swine farm. Therefore, preclinical identification of PEDV is of great significance for preventing the outbreak and spread of this disease. In this study, a functionalized nanoparticles-based PCR method (UNDP-PCR) specific for PEDV was developed through systematic optimization of functionalized magnetic beads and gold nanoparticles which were further used to specifically enrich viral RNA from the lysate of PEDV stool samples, forming a MMPs-RNA-AuNPs complex. Then, oligonucleotides specific for PEDV coated on AuNPs were eluted from the complex and were further amplified and characterized by PCR. The detection limitation of the established UNDP-PCR method for PEDV was 25 copies in per gram PEDV stool samples, which is 400-fold more sensitive than conventional RT-PCR for stool samples. The UNDP-PCR for PEDV exhibited reliable reproducibility and high specificity, no cross-reaction was observed with other porcine viruses. In 153 preclinical fecal samples, the positive detection rate of UNDP-PCR specific for PEDV (30.72%) was much higher than that of conventional RT-PCR (5.88%) and SYBR Green real-time RT-PCR. In a word, this study provided a RNA extraction and transcription free, rapid and economical method for preclinical PEDV infection, which showed higher sensitivity, specificity and reproducibility, and exhibited application potency for evaluating viral loads of preclinical samples.

Efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs

“…We hypothesized that this 25 aa deletion reduced the duration of the replication cycle of QIAP1401-p70. This finding is supported by previous reports that the 5' two-thirds of the coronavirus genome contains ORF1a and ORF 1b, which encode the viral RNA replicase [ 24 25 ].…”

Establishment of method for dual simultaneous detection of PEDV and TGEV by combination of magnetic micro-particles and nanoparticles

“…In our previous research, we have separately established detection methods for PEDV or TGEV. The principle of magnetic particle enrichment and specific nanoprobe amplification has achieved a strong specificity and convenient operation [12,13]. However, it is inconvenient to detect and distinguish these two viruses at same time using our previous PEDV or TGEV specific detection method.…”

Development of a SYBR green I-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3

“…To date, several approaches have been developed for the laboratory diagnosis of PEDV, including immunohistochemistry [46], enzymelinked immunosorbent assay (ELISA) [47], RT-PCR [48], reverse transcription loop-mediated isothermal amplification (RT-LAMP) [49], a nanoparticle-assisted PCR assay [50,51], and real-time RT-PCR [35,36]. Similarly, immunohistochemical detection, PCR, and qPCR have all been developed for the detection of PCV3 [20,33,34,37].…”