Establishment Of UNDP-PCR Assay For Rapid Detection Of TGEV And PEDV In Early Infection

With the rapid development of pig raising industry, porcine viral diarrhea has become one of the main epidemic diseases. Currently, porcine enteric epidemic diseases maily include transmissible gastroenteritis and porcine epidemic diarrhea. Commonly used detection methods are unable to detect these pathogens in early infection, thus lacking effective monitoring measures of these diseases and causing regional erupting prevalence. In addition, TGE and PED show great similarities in epidemiology, clinical symptoms and necropsy, so it is necessary to definitely identify them using laboratory tests. Therefore, establishing ultrasensitive differential detection methods of TGEV and PEDV has great significance for comprehensive control of porcine viral diseases. In this study, UNDP-PCR for PEDV and TGEV were developed respectively. Through systematical selection of PEDV and TGEV specific probes, optimal specific probes were coated to magnetic beads and gold nanoparticles respectively, followed by enriching viral nucleic acids from blood or feces and forming a sandwich-like complex MMPs-RNA-AuNPs, therefore the weak viral signals are fully amplified. Then DNA barcodes were characterized by PCR using specific primers. Based on above studies, a duplex UNDP-PCR method for TGEV and PEDV were also developed. The following results were obtained in this study:1. Establishment of TGEV UNDP-PCR for detecting TGEV from blood samples. On the basis of highly conserved region of TGEV ORF1 a, six specific probes were designed and coated to magnetic beads to prepare functional magnetic beads MMPs-p1 to p6, respectively. Functionalized MMPs-p1 to p6 were incubated in hybridization buffer at 40 oC for 30 minutes to form MMPs-RNA complexes, followed by TGEV specific RT-PCR. Next, SH-modified oligonucleotides(Oligos) 1-6 sharing same nucleic acid sequences of probe 1-6 were coated to gold nanoparticles(AuNPs) to prepare AuNPs-Oligo1 to Oligo6. Then PEDV RNA was incubated with oligos-functionalized AuNPs at 50 oC for 40 minutes. The formed complexes were then centrifuged and detected by PEDV specific RT-PCR. The results showed that p1 and Oligo2 exhibit higher specificity and capture ability for TGEV RNA and are optimal for amplification of viral signals, therefore MMPs-p1 and AuNPs-Oligo2 are used to establish TGEV UNDP-PCR. The results showed that the detection limit of this assay was 20 copies/mL, indicating that its sensitivity was 250-fold that of the conventional RT-PCR. In addition, this assay had high specificity for TGEV and did not show cross-reactivity other tested porcine viruses.2. Based on TGEV UNDP-PCR method, through optimization and improvement, PEDV UNDP-PCR was developed, which could detect PEDV from large-scale feces. Firstly, probes and oligos specific for PEDV were designed according to highly conserved regions of ORF1 a. Relying on strict selecting principles, 16 PEDV specific primers were designed, followed by probes selection. Then two selected optimal probes were coated to MMPs and AuNPs respectively, which were used to enrich PEDV RNA specificly and effectively. The results showed that the detection limitation of this assay was 25 copies in per gram PEDV stool samples, which is 400-fold more sensitive than conventional RT-PCR for stool sample. In addition, this assay exhibited a high level of specificity and reproducibility. In 153 preclinical fecal samples, the positive detection rate of UNDP-PCR specific for PEDV(30.72%) was more sensitive than that of conventional RT-PCR(5.88%).3. Establishment of duplex ultrasensitive nanoparticle DNA probe-based PCR assay(duplex UNDP-PCR) which was able to simultaneously detect TGEV and PEDV in the same reaction system. The results showed that the duplex UNDP-PCR was able to detect 25 copies each of TGEV and PEDV in feces, showing approximately 400-fold more sensitivity than conventional duplex RT-PCR assays. No cross-reaction was observed with other viruses.In conclusion, compared with other traditional detection techniques, the established TGEV UNDP-PCR, PEDV UNDP-PCR and duplex UNDP-PCR for TGEV and PEDV are time-saving and cost-effective methods with higher sensitivity, specificity and reproducibility, which could effectively control the occurrence, spread and massive outbreaks of pathogens before clinical pathological changes.