Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

Huang, Yong; Xing, Na; Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

doi:10.1371/journal.pone.0141545

Cited by 13 publications

(14 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Elution buffer (0.5 M DTT, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was used for release of thiolated DNA barcodes from the surface of functionalized AuNPs. The eluted DNA barcodes were purified and precipitated with NaAc and absolute alcohol, then were mixed with specific capture single strand DNA (ssDNA), followed by conventional PCR assay using PEDV specific detect-PCR primers ( Table 1) as described in the previous study [33,35]. The amplification products were separated on 1.5% agarose gel stained with ethidium bromide (EB) and visualized under ultraviolet (UV) light.…”

Section: Procedures Of Undp-pcr For Detection Of Pedvmentioning

confidence: 99%

“…The PCR products with the size of 451 bp were purified using gel extraction kit and cloned into pMD19-T vector (TAKARA, Japan) to construct the standard plasmid. Then the constructed plasmid was sequenced by Genscript (Nanjing, China).The plasmid concentration was determined by Nanodrop 2000 Spectrophotometer (Thermo scientific, USA), then the plasmid copy number was calculated as described in the previous study [33,35]. To test the specificity of the UNDP-PCR for PEDV, other viruses including PRRSV, TGEV, PCV2, PPV and CSFV were added to the reaction system and tested.…”

Section: The Specificity Sensitivity and Reproducibility Of The Undpmentioning

confidence: 99%

“…To find an economical and rapid diagnostic method with higher specificity, sensitivity and reproducibility for PEDV preclinical infection, relevant experiments were implemented to establish a systematic and optimal protocol for UNDP-PCR assay for PEDV, as schematically depicted in Fig 1. In our previous study, we have optimized a UNDP-PCR assay for TGEV in which the probes and oligos were targeted to replicase protein-encoding region ORF1a [35]. Quite similar to TGEV in genetic structure, the 5' two-thirds of PEDV genome encodes two polyproteins (1a and 1b) necessary for RNA replication and only exists in genomic RNA (gRNA).…”

Section: Probes Design and Optimization Of Undp-pcr Assay For Detectimentioning

confidence: 99%

See 2 more Smart Citations

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

Xing

Guan

et al. 2016

PLoS ONE

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Procedures Of Undp-pcr For Detection Of Pedvmentioning

confidence: 99%

Section: The Specificity Sensitivity and Reproducibility Of The Undpmentioning

confidence: 99%

Section: Probes Design and Optimization Of Undp-pcr Assay For Detectimentioning

confidence: 99%

See 1 more Smart Citation

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

Xing

Guan

et al. 2016

PLoS ONE

Self Cite

View full text Add to dashboard Cite

show abstract

“…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.…”

mentioning

confidence: 99%

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

Luo

Chen

et al. 2020

Journal of Infection and Chemotherapy

Self Cite

View full text Add to dashboard Cite

a b s t r a c tTransmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the main pathogens causing viral diarrhea in pig, mixed infections of these two viruses are very common in intensive pig rearing. However, there is a lack of a method to simultaneously detect and distinguish PEDV and TGEV in preclinical levels. In this study, we aimed to establish a dual ultrasensitive nanoparticle DNA probe-based PCR assay (dual UNDP-PCR) based on functionalized magnetic bead enrichment and specific nano-technology amplification for simultaneous detection and distinguish diagnosis of PEDV and TGEV. The detection limit of dual UNDP-PCR for single or multiple infections of PEDV and TGEV is 25 copies/g, which is 400 times more sensitive than the currently known duplex RT-PCR, showing better specificity and sensitivity without cross-reaction with other viruses. For pre-clinical fecal samples, the dual UNDP-PCR showed a markedly higher positive detection rate (52.08%) than conventional duplex RT-PCR (13.21%), can rapidly and accurately identify targeted pathogens whenever simple virus infection or co-infection. In summary, this study provides a technique for detecting and distinguishing PEDV and TGEV in preclinical levels, which is high sensitivity, specificity, repeatability, low cost and broad application prospect.

show abstract

“…EU366323) used in this study were isolated and purified previously by our team and stocked in our laboratory, the UV-inactivation was performed by UV radiation of the virus for 45 min in the hood. The anti-PCV2 Cap primary antibodies were produced by our team [12, 13]. The primary monoclonal rabbit antibodies of p53, p21 and anti-BrdU were purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

p53 signaling modulation of cell cycle arrest and viral replication in porcine circovirus type 2 infection cells

Han

et al. 2016

Vet Res

Self Cite

View full text Add to dashboard Cite

Porcine circovirus type 2 (PCV2) is a ubiquitous pathogen in the swine industry worldwide. Previous studies have shown that PCV2 infection induces host cell apoptosis through up-regulation of p53. To further identify the regulatory roles of p53 signaling in the process of PCV2 infection, we established p53 gene knockout PK15 cell lines using the genomic editor tool CRISPR/Cas9, and further investigated the roles of p53 in modulating the cell cycle and viral replication in this study. The results show that PCV2 infection induced obvious S phase accumulation in wild-type PK15 cells and a compromised S phase accumulation in the p53 gene mutation cells (813PK15p53m/m), but did not induce obvious S phase accumulation in the p53 gene knockout cells (148PK15p53−/−) compared with the respective mock infection. PCV2 infection activated p53 signaling, up-regulated the expression of p21, Cyclin E, and down-regulated Cyclin A, CDK2. In p53 deficient cells, however, PCV2-induced changes in Cyclin A, CDK2, and Cyclin E were efficiently reversed to the basal levels. Detection of PCV2 replication showed decreased viral ORF1 genomic DNA in p53 deficient cells (148PK15p533−/−) and p53 mutated cells (813PK15p53m/m) compared with p53 wild-type cells after different synchronization treatment. Furthermore, PCV2 viral genomic DNA and Cap protein levels were higher in the cells released from S phase synchronized cells than in the cells released from the G0/G1 phase or G2/M phase-synchronized, or asynchronous cells after 18 h post-infection. Taken together, this study demonstrates that PCV2 infection induces S phase accumulation to favor viral replication in host cells through activation of the p53 pathway.

show abstract

Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

Cited by 13 publications

References 30 publications

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

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

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

p53 signaling modulation of cell cycle arrest and viral replication in porcine circovirus type 2 infection cells

Contact Info

Product

Resources

About