Development of a nanoparticle-assisted PCR assay for detection of porcine epidemic diarrhea virus (original) (raw)
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AMB Express
A simple colorimetric assay is presented for detecting bovine viral diarrhea virus (BVDV)-RNA based on aggregation of gold nanoparticles (AuNPs) in the presence of charge-neutral peptide nucleic acids (PNA). Free charge-neutral PNA oligomers tended to be adsorbed onto AuNPs and act as a coagulant, whereas hybridizing complementary RNA with PNA disrupted PNA-induced AuNP aggregation, and the NPs remained stable. However, non-complementary RNA did not have this effect, and PNA induced aggregation of the AuNPs that resulted in a color change of the reaction from red to blue. The label-free colorimetric assay developed was estimated to have a 10.48 ng/reaction BVDV-RNA detection limit for the visual assay and 1.05 ng/reaction BVDV-RNA using a spectrophotometer. Diagnostic sensitivity and specificity for the assay was in accordance with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and nested RT-PCR results were 98 and 100%, respectively. Absorption of the 520/620 nm ratio was linear, along with an increase in the target RNA concentration of 1.64-52.4 ng/reaction (R 2 = 0.992), which showed a linear correlation for the quantitative assay. This study established a rapid visual label and enzyme-free diagnostic assay for detecting BVDV that is applicable in any clinical laboratory.
2021
Background Bovine viral diarrhea virus (BVDV) is a major economic disease that has been spread in most countries. In addition to vaccination, one of the main ways to control the disease and prevent it from spreading is to detect and cull infected animals, especially those with persistent infection (PI). We developed and compared two colorimetric biosensor assays based on probe-modified gold nanoparticles (AuNPs) to detect BVDV. Specific probes were designed to detect the 5′ untranslated region of BVDV-RNA. The thiolated probes were immobilized on the surface of the AuNPs. Two methods of cross-linking (CL) and non-crosslinking (NCL) probe-AuNPs hybridization were developed and compared. Results The hybridization of positive targets with the two probe-AuNPs formed a polymeric network between the AuNPs which led to the aggregation of nanoparticles and color change from red to blue. Alternatively, in the NCL mode, the hybridization of complementary targets with the probe-AuNPs resulted ...
Biology
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 te...
Virology journal, 2015
Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine. Recently, the outbreak of the epidemic disease has been a serious problem in swine industry. The objective of this study is to develop a rapid, sensitive, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of porcine epidemic diarrhea virus (PEDV) in less equipped laboratories. The optimal reaction condition of the current real-time RT-LAMP for PEDV was 62 °C for 45 min. It was capable of detecting PEDV from clinical samples and differentiating PEDV from several related porcine viruses, while it did not require additional expensive equipment. The minimum detection limit of the real-time RT-LAMP assay was 0.07PFU per reaction for PEDV RNA, making this assay approximately 100-fold more sensitive than that of one-step RT-PCR. By screening a panel of clinical specimens, the r...
Frontiers in Microbiology, 2020
Coronaviruses (CoVs) cause severe respiratory, enteric, and systemic infections in a wide range of hosts, including humans and animals. Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of porcine epidemic diarrhea (PED), a highly contagious intestinal disease affecting pigs of all ages. In this study, we optimized a viability real-time reverse transcriptase polymerase chain reaction (RT-qPCR) for the selective detection of infectious and heat-inactivated PEDV. PEMAX ™ , EMA ™ , and PMAxx ™ photoactivable dyes along with PtCl 4 and CDDP platinum compounds were screened as viability markers using two RT-qPCR assays: firstly, on PEDV purified RNA, and secondly on infectious and thermally inactivated virus suspensions. Furthermore, PMAxx ™ pretreatment matched the thermal inactivation pattern obtained by cell culture better than other viability markers. Finally, we further optimized the pretreatment by coupling viability markers with Triton X-100 in inoculated serum resulting in a better estimation of PEDV infectivity than RT-qPCR alone. Our study has provided a rapid analytical tool based on viability RT-qPCR to infer PEDV infectivity with potential application for feed and feed ingredients monitoring in swine industry. This development would allow for greater accuracy in epidemiological surveys and outbreak investigations.
Current Issues in Molecular Biology
Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detectio...
Virus Research, 2016
Porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute, highly contagious, and severe enteric disease that leads to high mortality rates in suckling piglets. Therefore, rapid diagnosis of PEDV infection is critical for the implementation of control measures for the virus. Many diagnostic tests have been recently developed and are currently available for the detection of PEDV, its proteins or nucleic acid, including virus isolation, immunofluorescence (IF) or immunohistochemistry (IHC), polymerase chain reaction (PCR) and isothermal amplification assays. Additionally, several serological assays have been developed and are currently used for the detection of antibodies against PEDV. Molecular assays such as real-time reverse transcriptase-PCR (rRT-PCR) became the methods of choice for the diagnosis of PEDV infection, providing sensitive, specific and rapid detection of the virus RNA in clinical samples. Whereas serological assays have been widely used to monitor prior exposure to the virus and to evaluate the efficacy of novel vaccine candidates or vaccination strategies. Here we discuss the properties of current PEDV diagnostic assays and prospects for improving diagnostic strategies in the future.
Journal of Virological Methods, 2009
A one-step real-time RT-PCR method has been developed for the simultaneous detection of both genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). The assay is based on primer-probe energy transfer, and the most important advantage of this is the relative tolerance towards mutations in the target-probe region. The primers and the probe were designed using an alignment of 235 Type 1 (including all subtypes) and Type 2 PRRSV strains. According to the alignment, multiple degenerations were included in the forward and reverse primers to enable the detection of all PRRSV strains deposited in the GenBank. Specificity was tested using 37 different PRRSV strains and eight other swine pathogen viruses. The detection limit was approximately 10 copies of RNA prepared from the Lelystad virus, a European Subtype 3 virus (Belarus strain Soz-8), and an American vaccine virus (Ingelvac MLV ® ). One TCID 50 was the detection limit in the case of the cell cultured Lelystad virus and an American wild type isolate, respectively. The melting point analysis revealed melting point decrease, but no significant sensitivity and signal loss in the presence of numerous (up to five) target-probe mismatches, indicating the capability of tolerating even more mutations. The method was suitable for the detection and quantitation of phylogenetically divergent strains and can serve as a robust, high throughput tool for molecular diagnosis of the PRRSV.
Research in Veterinary Science
Porcine epidemic diarrhoea virus (PEDV), belongs to the genus Alphacoronavirus in the family Coronaviridae and causes severe diarrhoea, vomiting, dehydration and high mortality in seronegative newborn piglets. Thus, a precise and rapid diagnosis of PEDV infection is important for the application of control measures to limit viral dissemination. In this investigation, a monoclonal antibodies (MAbs)-based competitive enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against PEDV was developed and validated. The diagnostic performance of the test was evaluated by receiver operating characteristic (ROC) analysis using a panel of 829 known sera collected from different commercial pig farms, with or without a history of PED presence and from experimentally infected pigs. The competitive ELISA showed excellent diagnostic performance and discriminatory power with high sensitivity (Se) and specificity (Sp) values (Se = 96.5%, 95% IC 94.1-98.1; Sp = 98.2%, 95% IC 96.3-99.3). Moreover, this competitive ELISA method has other properties, such as high feasibility of testing sera without pre-treatment and automatic and instrument-mediated revealing, that makes it appropriate for large-scale screenings of affected pig farms in endemic regions or for monitoring plans in PEDVfree areas.