Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus

Zhou, Xinrong; Zhang, Tiansheng; Song, Deping; Huang, Tao; Peng, Qi; Chen, Yanjun; Li, Anqi; Zhang, Fanfan; Wu, Qiong; Ye, Yu; Tang, Yuxin

doi:10.1016/j.mcp.2017.02.002

Cited by 58 publications

(59 citation statements)

References 24 publications

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“…The relative expression levels of the cytokines were compared with those of β-actin and uninfected piglets, using the 2 −∆∆Ct method. Specific primers for TLR-4, -9, TGF-β, TNF-α, β-actin, and PEDV-M sequences are shown in Table 1 [17][18][19]. All piglets were tested and for each piglet, three samples were tested.…”

Section: Real-time Rt-pcr (Qrt-pcr) Analysismentioning

confidence: 99%

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Oral Immunization against PEDV with Recombinant Lactobacillus casei Expressing Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV in Piglets

Hou

Jiang

Jin

et al. 2018

Viruses

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Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets. In our previous study, a genetically engineered Lactobacillus casei oral vaccine (pPG-COE-DCpep/L393) expressing a dendritic cell (DC)-targeting peptide fused with porcine epidemic diarrhea virus (PEDV) COE antigen was developed. This vaccine induced significant levels of anti-PEDV specific IgG and IgA antibody responses in mice, indicating a potential strategy against PEDV infection. In this study, pPG-COE-DCpep/L393 was used for oral vaccination of newborn piglets against PEDV. We then assessed the immune responses and protection efficacy of pPG-COE-DCpep/L393. An indirect enzyme-linked immunosorbent assay (ELISA) showed that the recombinant Lactobacillus vaccine elicits a specific systemic and mucosal immune response. The T-helper cells mediated by pPG-COE-DCpep/L393 and PEDV infection display a Th1 phenotype. The histopathological results showed that pPG-COE-DCpep/L393 promotes lymphocyte proliferation and effectively protects piglets against PEDV infection. The transforming growth factor-β level indicated that the recombinant Lactobacillus vaccine plays a role in anti-inflammatory responses in mesenteric lymph nodes during PEDV infection. These results show that pPG-COE-DCpep/L393 is a potential vaccine against PEDV infection.

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Section: Real-time Rt-pcr (Qrt-pcr) Analysismentioning

confidence: 99%

“…Viruses 2018, 10, x 5 of 14 α, β-actin, and PEDV-M sequences are shown in Table 1 [17][18][19]. All piglets were tested and for each piglet, three samples were tested.…”

Section: Production Of Mucosal and Humoral Immunitymentioning

confidence: 99%

Oral Immunization against PEDV with Recombinant Lactobacillus casei Expressing Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV in Piglets

Hou

Jiang

Jin

et al. 2018

Viruses

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“…At present, numerous established laboratory diagnostic techniques are used to identify PEDV. Kim [21] compared PEDV detection using RT-PCR, immunohistochemistry, and in situ hybridization; Zhou [22] developed and evaluated three assays, including conventional RT-PCR, SYBR Green I real-time RT-PCR, and TaqMan real-time RT-PCR assays. Their results indicated that the TaqMan real-time RT-PCR could be a useful tool for clinical diagnosis, epidemiological surveys, and outbreak investigations of PEDV.…”

Section: Discussionmentioning

confidence: 99%

Visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction method

Wang

et al. 2019

BMC Vet Res

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Background Porcine epidemic diarrhea virus (PEDV) is a major etiological agent of porcine epidemic diarrhea around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The aim of this study was to establish a novel reverse transcription polymerase spiral reaction (RT-PSR) assay for the rapid detection of porcine epidemic diarrhea virus (PEDV). For the assay, a specific RT-PSR primer pair was designed against a conserved region in PEDV ORF3. Results The RT-PSR was optimized, and PEDV could be detected after a 50 min incubation at 62 °C, in addition to the 15 min required for reverse transcription. No cross-reaction with other porcine infectious viruses was observed. This new method for PEDV detection was 10 times more sensitive than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay. The positive rates for 65 clinical samples using the new RT-PSR assay and the conventional RT-PCR assay were 58.46% (38/65) and 53.84% (35/65), respectively. In the RT-PSR assay, the addition of a mixture of dyes allowed a positive reaction to be directly observed by the naked eye. Conclusions These results indicate that this RT-PSR assay is capable of accurately detecting PEDV, and has the advantages of high specificity and sensitivity for the detection of PEDV.

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“…PCV3-positive and PEDV-positive clinical samples were used to optimize the duplex qPCR conditions. The positive tissue samples were collected from pig farms and identified as positive for PCV3 or PEDV using previously described qPCR assays [33,38] and sequencing. A total of 66 samples (intestinal tissues and fecal) were collected from 66 piglets suffering from diarrhea at different pig farms in Hebei province and Henan province (China) from 2014 to 2018.…”

Section: Virus and Clinical Samplesmentioning

confidence: 99%

“…Sixty-six clinical samples (the intestinal tissues and fecal) obtained from 66 piglets with diarrhea were tested using the established one-step duplex RT-qPCR method. For clinical samples, single qPCR and duplex qPCR methods were performed as the above described methods using the DNA or RNA from the samples, and compared with the results of previously reported qPCR assays for PEDV [38] and PCV3 [33]. The detection rates of the qPCR methods were compared, and all of the positive clinical samples detected by the duplex qPCR assay were confirmed by DNA sequencing.…”

Section: Application To Clinical Samplesmentioning

confidence: 99%

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

Han

Zheng

Zhao

et al. 2019

Molecular and Cellular Probes

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A B S T R A C TThe development of a rapid, specific, and sensitive SYBR Green I-based duplex real-time quantitative PCR assay is described for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (PCV3). The assay specifically detected PEDV and PCV3, with no fluorescence detected for other nontargeted pig pathogens. The assay showed a good linear relationship, and the limits of detection for this assay were 34.6 copies/μL and 61.2 copies/μL for PEDV and PCV3, respectively. The assay exhibited high repeatability and reproducibility, with intra-assay and inter-assay variation coefficients less than 2.0%. A clinical evaluation using intestinal tissue and fecal samples from piglets suffering from diarrhea at different pig farms in China revealed that the singular infection rates of PEDV and PCV3 were 43.94% (29/66) and 16.67% (11/66), respectively, while the co-infection rate of PCV3 with PEDV was 27.27% (18/66). The results indicate this assay is a rapid and reliable diagnostic tool for PEDV and PCV3 monitoring and surveillance in the field, and provides technical support for the quantitative detection of clinical samples infected or co-infected with PEDV and PCV3.

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Comparison and evaluation of conventional RT-PCR, SYBR green I and TaqMan real-time RT-PCR assays for the detection of porcine epidemic diarrhea virus

Cited by 58 publications

References 24 publications

Oral Immunization against PEDV with Recombinant Lactobacillus casei Expressing Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV in Piglets

Oral Immunization against PEDV with Recombinant Lactobacillus casei Expressing Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV in Piglets

Visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction 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

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