Molecular Detection of Feline Coronavirus Based on Recombinase Polymerase Amplification Assay

Kobialka, Rea Maja; Ceruti, Arianna; Bergmann, Michelle; Hartmann, Katrin; Truyen, Uwe; Wahed, Ahmed Abd El

doi:10.3390/pathogens10101237

Cited by 7 publications

(9 citation statements)

References 56 publications

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“…In contrast, molecular methods require few hours in case of real-time RT-PCR or 15 minutes for RT-RAA. The real-time RT-PCR must be performed using an expensive thermal cycler, while the RT-RAA was conducted in a mobile suitcase lab [ 21 , 36 ].…”

Section: Discussionmentioning

confidence: 99%

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Validation of the efficacy of air purifiers using molecular techniques

et al. 2023

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The importance of air purifiers has increased in recent years, especially with the “coronavirus disease 2019” pandemic. The efficacy of air purifiers is usually determined under laboratory conditions before widespread application. The standard procedure for testing depends on virus cultivation and titration on cell culture. This, however, requires several days to deliver results. The aim of this study was to establish a rapid molecular assay which can differentiate between intact infectious and distorted non-infectious virus particles. Feline Coronavirus was selected as model for screening. First the samples were pretreated with enzymes (universal nuclease and RNase cocktail enzyme mixture) or viability dye (propidium monoazide) to eliminate any free nucleic acids. The ribonucleic acid (RNA) from intact virus was released via magnetic beads-based extraction, then the amount of the RNA was determined using real-time reverse transcription polymerase chain reaction (RT-PCR) or reverse transcription recombinase-aided amplification (RT-RAA). All results were compared to the infectivity assay based on the calculation of the 50% tissue culture infectious dose (TCID50). The nuclease has eliminated 100% of the free Feline Coronavirus RNA, while propidium monoazide underperformed (2.3-fold decrease in free RNA). Both RT-RAA and real-time RT-PCR produced similar results to the infectivity assay on cell culture with limit of detection of 102 TCID50/mL. Two UV-C air purifiers with prosperities of 100% inactivation of the viruses were used to validate the established procedure. Both real-time RT-PCR and RT-RAA were able to differentiate between intact virus particles and free RNA. To conclude, this study revealed a promising rapid method to validate the efficacy of air purifiers by combining enzymatic pretreatment and molecular assays.

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Section: Discussionmentioning

confidence: 99%

“…A previously published protocol for the detection of a highly conserved region of the FCoV 7b gene was used for real-time RT-RAA [ 36 ]. RT-RAA Nucleic Acid Amplification Kit (Fluorescent Method, Jiangsu Qitian Gene Technology Co., Ningbo, China) was used.…”

Section: Methodsmentioning

confidence: 99%

Validation of the efficacy of air purifiers using molecular techniques

et al. 2023

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“…However, FCoV RNA can also be detected outside of the gastrointestinal tract in cats without FIP infection [24]. A reverse transcriptionrecombinase polymerase amplifcation (RT-RPA) method was used for rapid FCoV detection [25]. RT-RPA has been used to detect the nucleic acid sequences from a wide variety of pathogens within a 15-min assay run-time [25,26].…”

Section: Introductionmentioning

confidence: 99%

“…A reverse transcriptionrecombinase polymerase amplifcation (RT-RPA) method was used for rapid FCoV detection [25]. RT-RPA has been used to detect the nucleic acid sequences from a wide variety of pathogens within a 15-min assay run-time [25,26]. Te rapid results were achieved using additional proteins to separate the DNA strands instead of thermal cycling as in the PCR [25].…”

Section: Introductionmentioning

confidence: 99%

A Combined Method Based on the FIPV N Monoclonal Antibody Immunofluorescence Assay and RT-nPCR Method for the Rapid Diagnosis of FIP-Suspected Ascites

Ding

et al. 2023

Transboundary and Emerging Diseases

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Feline infectious peritonitis (FIP), which is caused by feline infectious peritonitis virus (FIPV), is a fatal and immunologically mediated infectious disease among cats. At present, due to the atypical clinical symptoms and clinicopathological changes, the clinical diagnosis of FIP is still difficult. The gold standard method for the differential diagnosis of FIP is immunohistochemistry (IHC) which is time-consuming and requires specialized personnel and equipment. Therefore, a rapid and accurate clinical diagnostic method for FIPV infection is still urgently needed. In this study, based on the etiological investigation of FIPV in parts of southern China, we attempted to explore a new rapid and highly sensitive method for clinical diagnosis. The results of the etiological investigation showed that the N gene of the FIPV BS8 strain had the highest homology with other strains. Based on this, a specific FIPV BS8 N protein monoclonal antibody was successfully prepared by expression of the recombinant proteins, immunization of mice, fusion and selection of hybridoma cell lines, and screening and purification of monoclonal antibodies. Furthermore, we carried out a time-saving combination method including indirect immunofluorescence assay (IFA) and nested reverse transcription polymerase chain reaction (RT-nPCR) to examine FIP-suspected clinical samples. These results were 100% consistent with IHC. The results revealed that the combined method could be a rapid and accurate application in the diagnosis of suspected FIPV infection within 24 hours. In conclusion, the combination of IFA and RT-nPCR was shown to be a fast and reliable method for clinical FIPV diagnosis. This study will provide insight into the exploitation of FIPV N antibodies for the clinical diagnosis of FIP-suspected ascites samples.

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“…The entire amplification process can be completed within 20 min at 37°C-42°C (Salazar et al, 2021), which makes RPA a promising on-site detection method. Currently, RPA is successfully employed for the molecular detection of diverse pathogens, such as bacteria, fungi, parasites, and viruses, with different detection strategies (Yang et al, 2020;Arif et al, 2021;Guo et al, 2021;Kobialka et al, 2021); however, there are no reports on the application of the RPA method for MHV. Hence, in this study, fluorescent probe-based RT-RPA assay for MHV detection was developed based on the highly conserved region of the M gene.…”

Section: Introductionmentioning

confidence: 99%

Real-time reverse transcription recombinase polymerase amplification for rapid detection of murine hepatitis virus

Wang¹,

Sui²,

et al. 2022

Front. Microbiol.

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Murine hepatitis virus (MHV) is a highly infectious murine coronavirus that has a high potential for causing harm to host animals. This study aimed to develop a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for rapid detection of MHV in laboratory mice.MethodsSpecific primers and probes for RT-RPA assay were designed targeting the conserved region in the M gene of the MHV reference strain (accession no. FJ6647223) according to the TwistDx manual instructions. The specificity, sensitivity, and reproducibility of the RT-RPA method were evaluated and compared with those of the standard RT-qPCR method. The clinical applicability of this assay was evaluated using 68 field samples.ResultsAmplification using the newly developed RT-RPA assay was completed within 20 min at 37°C, while that using the RT-qPCR method required nearly 60 min. The RT-RPA method exhibited an obvious time-saving advantage. Both RT-RPA and RT-PCR methods had the same limit of detection, which was 4.45 × 101 copies/μL. The specificity was indicated by a lack of cross-reaction with MHV, pneumonia virus of mice, Sendai virus, hantavirus, minute virus of mice, and reovirus type III. The MHV detection rate of RT-RPA assays was 13.63% (9/66) and RT-qPCR assays was 15.15% (10/66). Cohen’s “kappa” (κ) analysis results exhibited a very good agreement between two methods with the value of κ ≥ 0.750(since κ = 0.939) and p < 0.0005 (since p = 0.000).ConclusionThe RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of MHV in laboratory mice and has significant potential for application in laboratories.

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Molecular Detection of Feline Coronavirus Based on Recombinase Polymerase Amplification Assay

Cited by 7 publications

References 56 publications

Validation of the efficacy of air purifiers using molecular techniques

Validation of the efficacy of air purifiers using molecular techniques

A Combined Method Based on the FIPV N Monoclonal Antibody Immunofluorescence Assay and RT-nPCR Method for the Rapid Diagnosis of FIP-Suspected Ascites

Real-time reverse transcription recombinase polymerase amplification for rapid detection of murine hepatitis virus

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