Rapid detection of porcine deltacoronavirus and porcine epidemic diarrhea virus using the duplex recombinase polymerase amplification method

Li, Gen; Wu, Miaoli; Li, Jinhui; Cai, Weiyou; Xie, Yongsheng; Si, Guangbing; Xiao, Li; Feng, Cong; He, Dongsheng

doi:10.1016/j.jviromet.2021.114096

Cited by 10 publications

(7 citation statements)

References 29 publications

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“…Herein, in the 23 PEDV-positive clinical samples determined using RT-qPCR, 22 were positive for the clinical PEDV specimens by the RT-RPA-NALF assay. This new assay postulates higher sensitivity (95.65%) and specificity (100%) than those in previous research [ 33 , 34 , 35 ], which indicates the reliability and accuracy of this diagnostic test kit. Though the limitation of this RT-RPA-NALF assay was its inability to distinguish wild-type strains from the vaccine strain, the test results in this study and the aforementioned reports both indicate that the wild-type virus was commonly found to be the mainstream of clinical specimens, whereas the number of clinical samples containing the vaccine strains was quite low.…”

Section: Discussionmentioning

confidence: 57%

Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus

Pewlaoo

Phanthong

Kong-Ngoen

et al. 2022

Biology

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Porcine epidemic diarrhea virus (PEDV) infection is an important acute diarrheal disease of swine that results in economic and industrial losses worldwide. The clinical manifestations in infected piglets are severe diarrhea, dehydration with milk curd indigestion, leading to death. The diagnosis of PEDV is essential for monitoring and managing the disease. PEDV can be detected and identified by serology and the nucleic acid of the virus in clinical samples. Therefore, a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow (RT-RPA-NALF) was developed for the rapid detection of PEDV. Qualitative reverse transcription-polymerase chain reaction (RT-qPCR) was established as the gold standard assay to compare results. Specific primer pairs and probes were designed, and RT-RPA conditions were optimized to amplify the M gene of PEDV. The established RT-RPA-NALF assay could finish in 25 min at a temperature of 42 °C and the amplicon interpreted by visual detection. The developed RT-RPA-NALF assay was specific to the M gene of PEDV, did not detect other common swine diarrhea pathogens, and showed minimal detection at 102 TCID50/mL PEDV. The RT-RPA-NALF assay can detect PEDV in 5 simulated fecal samples. Furthermore, in 60 clinical fecal samples, the results of RT-RPA-NALF correlated with RT-qPCR assay, which provides sensitivity of 95.65% and specificity of 100%, with a coincident rate of 98.33%. The rapid RT-RPA-NALF is simple and rapid, increases high sensitivity, and can be used in the field.

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Section: Discussionmentioning

confidence: 57%

Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus

Pewlaoo

Phanthong

Kong-Ngoen

et al. 2022

Biology

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“…Alternate novel isothermal amplification techniques such as RT-LAMP and RT-RPA are rapid and do not require expensive equipment such as a thermal cycler, and hence can be performed in a point-of-care context or resource-poor laboratories. In our current meta-analysis, 7 of 13 studies with NAIA-based POCT used an RT-LAMP assay [ 29 , 30 , 33 , 35 , 37 , 40 , 41 ] and 6 studies adopted an RT-RPA assay to detect PEDV [ 31 , 32 , 34 , 36 , 38 , 39 ]. In general, the sensitivities of nucleic acid tests are in the order of real-time RT-PCR followed by RT-LAMP, RT-RPA, and conventional RT-PCR [ 53 ].…”

Section: Discussionmentioning

confidence: 99%

Point-of-Care Tests for Rapid Detection of Porcine Epidemic Diarrhea Virus: A Systematic Review and Meta-Analysis

Tian

Pang

et al. 2022

Viruses

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The timely and accurate diagnosis of porcine epidemic diarrhea virus (PEDV) infection is crucial to reduce the risk of viral transmission. Therefore, the objective of this review was to evaluate the overall diagnostic accuracy of rapid point-of-care tests (POCTs) for PEDV. Studies published before 7 January 2022 were identified by searching PubMed, EMBASE, Springer Link, and Web of Science databases, using subject headings or keywords related to point of care and rapid test diagnostic for PEDV and PED. Two investigators independently extracted data, rated risk of bias, and assessed the quality using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The bivariate model and the hierarchical summary receiver operating characteristic (HSROC) model were used for performing the meta-analysis. Threshold effect, subgroup analysis, and meta-regression were applied to explore heterogeneity. Of the 2908 records identified, 24 eligible studies involving 3264 specimens were enrolled in the meta-analysis, including 11 studies on evaluation of lateral flow immunochromatography assay (ICA)-based, and 13 on nucleic acid isothermal amplification (NAIA)-based POCTs. The overall pooled sensitivity, specificity and diagnostic odds ratio (DOR) were 0.95 (95% CI: 0.92–0.97), 0.96 (95% CI 0.88–0.99) and 480 (95% CI 111–2074), respectively; for ICA-based POCTs and the corresponding values for NAIA-based, POCTs were 0.97 (95% CI 0.94–0.99), 0.98 (95% CI 0.91–0.99) and 1517 (95% CI 290–7943), respectively. The two tests showed highly comparable and satisfactory diagnostic performance in clinical utility. These results support current recommendations for the use of rapid POC tests when PEDV is suspected.

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“…e ., it involves the selection of the target region, designing the primer candidates, and screening for the best candidate ( Lobato and O'Sullivan, 2018 ). Hairpin structure, primer dimers, and primer–primer interactions should be avoided ( Li et al., 2021 ). Nevertheless, we detected several mutations in the ORF2 gene sequence of HEV.…”

Section: Discussionmentioning

confidence: 99%

Rapid and direct detection of hepatitis E virus in raw pork livers by recombinase polymerase amplification assays

Wang

Wang²,

Zhou³

et al. 2022

Front. Cell. Infect. Microbiol.

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Hepatitis E virus (HEV) is a zoonotic pathogen that causes global hepatitis E. Outbreaks of hepatitis E are directly linked to the consumption of pork liver products. Herein reverse transcription recombinase polymerase amplification assays targeting the ORF2 gene were developed for the rapid detection of HEV by integrating the fluorescence detection platform (qRT-RPA) and the visible lateral flow biosensor by naked eyes (LFB RT-RPA). The qRT-RPA assay effectively detected HEV RNA with a limit of detection (LOD) of 154 copies/μl (95%CI: 126–333 copies/µl) in Genie III at 41°C for 20 min. Besides this, the LFB RT-RPA detected the HEV RNA with a LOD of 24 copies/μl (95%CI: 20–57 copies/µl) in an incubator block at 41°C for 20 min. The developed RT-RPA assays also showed good specificity for HEV, with no cross-reactions with any of the other important swine pathogens examined in this work. The performance of the developed RT-RPA assays was validated on 14 HEV RNA-positive and 66 HEV RNA-negative raw pork liver samples identified by a previously described qRT-PCR. Consequently, 11 and 12 samples were HEV RNA-positive as detected by the qRT-RPA and the LFB RT-RPA, respectively. Compared to qRT-PCR, the qRT-RPA and LFB RT- RPA assays revealed a coincidence rate of 96.3 and 97.5% as well as a Kappa value of 0.858 and 0.908, respectively. These results ascertain that the developed RT-RPA assays are effective diagnostic tools for the point-of-care detection of HEV in resource-limited settings.

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Rapid detection of porcine deltacoronavirus and porcine epidemic diarrhea virus using the duplex recombinase polymerase amplification method

Cited by 10 publications

References 29 publications

Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus

Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus

Point-of-Care Tests for Rapid Detection of Porcine Epidemic Diarrhea Virus: A Systematic Review and Meta-Analysis

Rapid and direct detection of hepatitis E virus in raw pork livers by recombinase polymerase amplification assays

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