{"title":"Development and Application of an RPA-Based Rapid Point-of-Care Testing (POCT) Method for the Detection of Feline Panleukopenia Virus","authors":"Liang Hong, Qian Huang, Yuhang Zhou, Qi Zheng, Shipeng Wang, Fangfang Chen, Xinyue Chang, Guosheng Jiang, Lisha Zha","doi":"10.1155/2024/3680778","DOIUrl":null,"url":null,"abstract":"<div>\n <p>Feline panleukopenia (FP) is a highly prevalent and consequential disease that poses a substantial threat to both adult and juvenile cats across all geographical regions. The causative agent responsible for this disease is the feline panleukopenia virus (FPV). Therefore, it is imperative to develop a facile, efficient, and accurate detection method for FPV. Hence, a recombinase polymerase amplification–lateral flow dipstick assay (RPA–LFDA) method was specifically designed for the detection of FPV. The amplification process was optimized. This investigation focused on evaluating the expansion temperature detection system and revealed an optimal reaction temperature of 39°C. Then, primer combination screening involving nine groups identified F3R2 as the most effective primer set, while dilution ratio experiments determined that a 10-fold dilution yielded the best amplification products. Our findings demonstrated that the RPA-LFDA assay had an analytical sensitivity that was capable of detecting as low as 10 target copies per reaction. Furthermore, cross-reactivity tests demonstrated no interference between feline herpesvirus-1 (FHV-1) and feline calicivirus (FCV). To validate our newly developed method against existing techniques in clinical samples from three common sources on the market, we observed superior sensitivity and specificity compared to those of the colloidal gold method (CGM), with a higher positive detection rate using our nucleic acid detection system than CGM. Compared to qPCR as a reference standard, RPA-LFDA detected 39 out of 44 positive samples (including one false positive), whereas CGM detected 26 out of 44 positive samples. Based on the RPA-LFDA, the sensitivity was calculated to be 100%, the specificity was 83.33%, the mistake diagnostic rate was 16.67%, the omission diagnostic rate was 0%, and the overall accuracy reached 97.73%. Moreover, the positive coincidence rate was 97.44%, while the negative coincidence rate reached 100%. The agreement <i>κ</i> value was 0.8962. In conclusion, this approach exhibits greater sensitivity than CGM and offers greater convenience and cost-effectiveness than the qPCR methodology, making it a viable option for the clinical detection of FPV.</p>\n </div>","PeriodicalId":234,"journal":{"name":"Transboundary and Emerging Diseases","volume":null,"pages":null},"PeriodicalIF":3.5000,"publicationDate":"2024-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1155/2024/3680778","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Transboundary and Emerging Diseases","FirstCategoryId":"97","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1155/2024/3680778","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"INFECTIOUS DISEASES","Score":null,"Total":0}
引用次数: 0
Abstract
Feline panleukopenia (FP) is a highly prevalent and consequential disease that poses a substantial threat to both adult and juvenile cats across all geographical regions. The causative agent responsible for this disease is the feline panleukopenia virus (FPV). Therefore, it is imperative to develop a facile, efficient, and accurate detection method for FPV. Hence, a recombinase polymerase amplification–lateral flow dipstick assay (RPA–LFDA) method was specifically designed for the detection of FPV. The amplification process was optimized. This investigation focused on evaluating the expansion temperature detection system and revealed an optimal reaction temperature of 39°C. Then, primer combination screening involving nine groups identified F3R2 as the most effective primer set, while dilution ratio experiments determined that a 10-fold dilution yielded the best amplification products. Our findings demonstrated that the RPA-LFDA assay had an analytical sensitivity that was capable of detecting as low as 10 target copies per reaction. Furthermore, cross-reactivity tests demonstrated no interference between feline herpesvirus-1 (FHV-1) and feline calicivirus (FCV). To validate our newly developed method against existing techniques in clinical samples from three common sources on the market, we observed superior sensitivity and specificity compared to those of the colloidal gold method (CGM), with a higher positive detection rate using our nucleic acid detection system than CGM. Compared to qPCR as a reference standard, RPA-LFDA detected 39 out of 44 positive samples (including one false positive), whereas CGM detected 26 out of 44 positive samples. Based on the RPA-LFDA, the sensitivity was calculated to be 100%, the specificity was 83.33%, the mistake diagnostic rate was 16.67%, the omission diagnostic rate was 0%, and the overall accuracy reached 97.73%. Moreover, the positive coincidence rate was 97.44%, while the negative coincidence rate reached 100%. The agreement κ value was 0.8962. In conclusion, this approach exhibits greater sensitivity than CGM and offers greater convenience and cost-effectiveness than the qPCR methodology, making it a viable option for the clinical detection of FPV.
期刊介绍：
Transboundary and Emerging Diseases brings together in one place the latest research on infectious diseases considered to hold the greatest economic threat to animals and humans worldwide. The journal provides a venue for global research on their diagnosis, prevention and management, and for papers on public health, pathogenesis, epidemiology, statistical modeling, diagnostics, biosecurity issues, genomics, vaccine development and rapid communication of new outbreaks. Papers should include timely research approaches using state-of-the-art technologies. The editors encourage papers adopting a science-based approach on socio-economic and environmental factors influencing the management of the bio-security threat posed by these diseases, including risk analysis and disease spread modeling. Preference will be given to communications focusing on novel science-based approaches to controlling transboundary and emerging diseases. The following topics are generally considered out-of-scope, but decisions are made on a case-by-case basis (for example, studies on cryptic wildlife populations, and those on potential species extinctions):
Pathogen discovery: a common pathogen newly recognised in a specific country, or a new pathogen or genetic sequence for which there is little context about — or insights regarding — its emergence or spread.
Prevalence estimation surveys and risk factor studies based on survey (rather than longitudinal) methodology, except when such studies are unique. Surveys of knowledge, attitudes and practices are within scope.
Diagnostic test development if not accompanied by robust sensitivity and specificity estimation from field studies.
Studies focused only on laboratory methods in which relevance to disease emergence and spread is not obvious or can not be inferred (“pure research” type studies).
Narrative literature reviews which do not generate new knowledge. Systematic and scoping reviews, and meta-analyses are within scope.
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