Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus

Wang, Leyi; Eggett, Therese E.; Lanka, Saraswathi; Fredrickson, Richard; Li, Ganwu; Zhang, Yan; Yoo, Dongwan; Bowman, Andrew S.

doi:10.1016/j.jviromet.2019.04.008

Cited by 7 publications

(8 citation statements)

References 11 publications

(19 reference statements)

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“…Recently, a variety of laboratory tools has emerged to solve the immediate problems for better detection of ASFV, such as serodiagnosis (immunoelectroosmophoresis, IEOP) test [21], antigen ELISA [22], fluorescent antibody test (FAT) [23], immunochromatography test strip (ICTS) [24]) and molecular tools (PCR, RPA [25], multiplex RT-PCR [26] and qPCR [27,28]). The OIE has recommended several ASFV detection methods, including virus isolation, fluorescence antibody test (FAT) and conventional PCR or real-time PCR [29] in 2018.…”

Section: Discussionmentioning

confidence: 99%

Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus

Zhang

Guang

et al. 2021

PLoS ONE

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African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.

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Section: Discussionmentioning

confidence: 99%

Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus

Zhang

Guang

et al. 2021

PLoS ONE

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“…Two micrograms of total RNA were reverse transcribed using a poly (T) primer and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). PHEV N gene RNA was detected as previously described using primer and probe sequences that targeted the nucleocapsid gene of PHEV [ 34 ]. A LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA) was used to perform qPCR.…”

Section: Methodsmentioning

confidence: 99%

“…A LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA) was used to perform qPCR. Thermo cycling amplification conditions were as follows: 50 °C for 30 min, 94 °C for 15 min then 45 cycles of 95 °C for 15 s, 58 °C for 60 s, and 72 °C for 10 s, as previously described [ 34 ].…”

Section: Methodsmentioning

confidence: 99%

Suppression of porcine hemagglutinating encephalomyelitis virus replication by resveratrol

Liu

Song

Liu

et al. 2022

Virol J

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Background Porcine hemagglutinating encephalomyelitis virus (PHEV), a member of the genus Betacoronavirus, is the causative agent of neurological disease in pigs. No effective therapeutics are currently available for PHEV infection. Resveratrol has been shown to exert neuroprotective and antiviral effects. Here resveratrol was investigated for its ability to inhibit PHEV replication in nerve cells and central nervous system tissues. Methods Anti-PHEV effect of resveratrol was evaluated using an in vitro cell-based PHEV infection model and employing a mouse PHEV infection model. The collected cells or tissues were used for quantitative PCR analysis, western blot analysis, or indirect immunofluorescence assay. The supernatants were collected to quantify viral loads by TCID50 assay in vitro. EC50 and CC50 were determined by dose–response experiments, and the ratio (EC50/CC50) was used as a selectivity index (SI) to measure the antiviral versus cytotoxic activity. Results Our results showed that resveratrol treatment reduced PHEV titer in a dose-dependent manner, with a 50% inhibition concentration of 6.24 μM. A reduction of > 70% of viral protein expression and mRNA copy number and a 19-fold reduction of virus titer were achieved when infected cells were treated with 10 µM resveratrol in a pre-treatment assay. Quantitative PCR analysis and TCID50 assay results revealed that the addition of 10 μM resveratrol to cells after adsorption of PHEV significantly reduced 56% PHEV mRNA copy number and eightfold virus titer. 10 µM resveratrol treatment reduced 46% PHEV mRNA copy number and fourfold virus titer in virus inactivation assay. Moreover, the in vivo data obtained in this work also demonstrated that resveratrol inhibited PHEV replication, and anti-PHEV activities of resveratrol treatment via intranasal installation displayed better than oral gavage. Conclusion These results indicated that resveratrol exerted antiviral effects under various drug treatment and virus infection conditions in vitro and holds promise as a treatment for PHEV infection in vivo.

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“…In addition, the 1,977 samples were also tested by the multiplex qRT-PCR developed by Wang et al for detection of PHEV (31), and Wu et al for detection of PRV, CSFV, and JEV (38). The positivity rates of PHEV, PRV, CSFV, and JEV by the developed assay in this study were compared with those of these two methods, and their coincidence rates were evaluated.…”

Section: Detection Of the Clinical Samplesmentioning

confidence: 99%

“…The real-time quantitative PCR/ RT-PCR (qPCR/qRT-PCR) has the advantages of high sensitivity, excellent specificity, high throughput, uneasy contamination, and convenient operation, and is widely used to detect viral nucleic acids (30). To date, the qPCR/qRT-PCR has been reported to detect PHEV (31), PRV (32,33), CSFV (34,35), and JEV (36,37), and the multiplex qRT-PCR has been reported to detect PHEV, PRV, CSFV, and/or JEV (38)(39)(40). However, no multiplex qRT-PCR to simultaneously detect and differentiate PHEV, PRV, CSFV, and JEV has been reported until now.…”

Section: Introductionmentioning

confidence: 99%

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV

Hu,

Feng,

Shi

et al. 2023

Front. Vet. Sci.

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Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5′ untranslated region (5’UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/μL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.

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Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus

Cited by 7 publications

References 11 publications

Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus

Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus

Suppression of porcine hemagglutinating encephalomyelitis virus replication by resveratrol

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV

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