A novel duplex TaqMan probe-based real-time RT-qPCR for detecting and differentiating classical and variant porcine epidemic diarrhea viruses

Su, Yu; Liu, Yunchao; Chen, Yumei; Xing, Guangxu; Huang, Hao; Wei, Qiang; Liang, Yue; Xie, Weitao; Li, Dongliang; Huang, Huimin; Deng, Ruiguang; Zhang, Gaiping

doi:10.1016/j.mcp.2017.10.003

Cited by 29 publications

(22 citation statements)

References 38 publications

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“…Real‐time fluorescence qPCR is a commonly used and reliable detection method for nucleic acids. There are 2 approaches that use qPCR: 1 based on fluorescent dyes and 1 that is targeted at specific fluorescent‐labeled DNA sequences, called probes . The invention of TaqMan probes has overcome the limitations of SYBR Green I, that is, its lack of specificity owing to its ability to bind to all DNA double‐stranded structures .…”

Section: Discussionmentioning

confidence: 99%

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

et al. 2019

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Novel diagnostic and prognostic biomarkers of cancers are needed to improve precision medicine. Circular RNAs act as important regulators in cancers at the transcriptional and posttranscriptional levels. The circular RNA circMAN1A2 is highly expressed in nasopharyngeal carcinoma according to our previous RNA sequencing data; however, the expression and functions of circMAN1A2 in cancers are still obscure. Therefore, in this study, we evaluated the expression of circMAN1A2 in the sera of patients with nasopharyngeal carcinoma and other malignant tumors and analyzed its correlations with clinical features and diagnostic values. The expression levels of circMAN1A2 were detected by quantitative real‐time PCR, and the correlations of clinical features with circMAN1A2 expression were analyzed by χ2 tests. Receiver operating characteristic curves were used to evaluate the clinical applications of circMAN1A2. The results showed that circMAN1A2 was upregulated in nasopharyngeal carcinoma, oral cancer, thyroid cancer, ovarian cancer, and lung cancer, with areas under the curves of 0.911, 0.779, 0.734, 0.694, and 0.645, respectively, indicating the good diagnostic value of circMAN1A2. Overall, our findings suggested that circMAN1A2 could be a serum biomarker for malignant tumors, providing important insights into diagnostic approaches for malignant tumors. Further studies are needed to elucidate the mechanisms of circMAN1A2 in the pathogenesis of cancer.

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Section: Discussionmentioning

confidence: 99%

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

et al. 2019

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“…TaqMan-probe-based real-time qPCR is a rapid, high specificity, high sensitivity, and great reproducibility detection tool for identification of viruses (Chang et al 2014). Among the seven open reading frames (ORFs) and four structural protein genes, S (spike), E (envelope), M (membrane), N (nucleocapsid) of coronaviruses (Su et al 2018), the M and N genes are highly conserved within the same antigenic group but less homologous between each of these four swine enteric coronaviruses (Yang et al 2019). To establish a high-specific multiplex real-time RT-qPCR for the detection and differential diagnosis of swine enteric coronaviruses, primer sets, and TaqMan probes were designed targeting the highly conserved regions of PEDV or PDCoV M genes and TGEV or PEAV N genes, based on bioinformatics analysis of each virus.…”

Section: Discussionmentioning

confidence: 99%

A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses

Huang

Chen

Yao

et al. 2019

Appl Microbiol Biotechnol

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Swine enteric coronaviruses are a group of most significant pathogens causing diarrhea in piglets with similar clinical symptoms and pathological changes. To develop a simple, rapid, accurate, and high-throughput detection method for diagnosis and differential diagnosis on swine enteric coronaviruses, specific primers and probes were designed based on the highly conserved regions of transmissible gastroenteritis virus (TGEV) N, porcine epidemic diarrhea virus (PEDV) M, porcine deltacoronavirus (PDCoV) M, and porcine enteric alphacoronavirus (PEAV) N genes respectively. A TaqMan-probe-based multiplex real-time RT-qPCR assay was developed and optimized to simultaneously detect these swine enteric coronaviruses. The results showed that the limit of detection can reach as low as 10 copies in singular real-time RT-qPCR assays and 100 copies in multiplex real-time RT-qPCR assay, with all correlation coefficients (R 2 ) at above 0.99, and the amplification efficiency at between 90 and 120%. This multiplex real-time RT-qPCR assay demonstrated high sensitivity, extreme specificity, and excellent repeatability. The multiplex real-time RT-qPCR assay was then employed to detect the swine enteric coronavirus from 354 field diarrheal samples. The results manifested that TGEVand PDCoV were the main pathogens in these samples, accompanied by co-infections. This well-established multiplex real-time RT-qPCR assay provided a rapid, efficient, specific, and sensitive tool for detection of swine enteric coronaviruses.

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“…Then, the virus and protein mixtures were washed away. Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions and the PEDV binding was represented by the copies of the PEDV nucleocapsid (N) gene using absolute quantitative PCR (The primers were listed in Table 1) [42].…”

Section: Primer Namementioning

confidence: 99%

Characterization of the interaction between recombinant porcine aminopeptidase N and spike glycoprotein of porcine epidemic diarrhea virus

Sun

Jiang

et al. 2018

International Journal of Biological Macromolecules

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Porcine epidemic diarrhea (PED) has caused huge economic losses to the global pork industry. Infection by its causative agent PED virus (PEDV), an Alpha-coronavirus, was previously proven to be mediated by its spike (S) glycoprotein and a cellular receptor porcine aminopeptidase N (pAPN). Interestingly, some recent studies have indicated that pAPN is not a functional receptor for PEDV. To date, there is a lack of a direct evidence for the interaction between pAPN and PEDV S protein in vitro. Here, we prepared pAPN ectodomain and the truncated variants of PEDV S protein in Drosophila S2 cells. These recombinant proteins were homogeneous after purification by metal-affinity and size-exclusion chromatography. We then assayed the purified target proteins through immunogenicity tests, PEDV binding interference assays, circular dichroism (CD) measurements, pAPN activity assay and structural determination, demonstrating that they were biologically functional. Finally, we characterized their interactions by gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and surface plasmon resonance (SPR) analyses. The results showed that their affinities were too low to form complexes, which suggest that pAPN may be controversial as the genuine receptor for PEDV. Therefore, further research needs to be carried out to elucidate the interaction between PEDV and its genuine receptor.

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A novel duplex TaqMan probe-based real-time RT-qPCR for detecting and differentiating classical and variant porcine epidemic diarrhea viruses

Cited by 29 publications

References 38 publications

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

circMAN1A2 could serve as a novel serum biomarker for malignant tumors

A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses

Characterization of the interaction between recombinant porcine aminopeptidase N and spike glycoprotein of porcine epidemic diarrhea virus

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