Establishment of a Rapid LAMP Assay for <i>Aeromonas hydrophila</i> and Comparison with the Application of qPCR
The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employ...
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MDPI AG
2023-07-01
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| Series: | Metabolites |
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| Online Access: | https://www.mdpi.com/2218-1989/13/7/841 |
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| author | Zihui Gao Chunhua Yang Xiaobo Zhang Bing Hu Huang Zhang Zhihong Zhang Wendong Kuang Qiuyue Zheng Jijuan Cao |
| author_facet | Zihui Gao Chunhua Yang Xiaobo Zhang Bing Hu Huang Zhang Zhihong Zhang Wendong Kuang Qiuyue Zheng Jijuan Cao |
| author_sort | Zihui Gao |
| collection | DOAJ |
| description | The development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employing Loop-mediated isothermal amplification (LAMP) of Aeromonas hydrophila was devised and compared to the conventional qPCR method documented by Rong Wang. Validation of the diagnostic assay was carried out using actual samples obtained from aquaculture fish. The findings revealed that based on the rapid detection of crude bacterial genomic DNA, the fluorescent LAMP assay possessed a lower limit of detection (LOD) of 0.559 ng/μL (0.315–1.693, 95% CI), while the LOD for qPCR stood at 4.301 ng/μL (2.084–8.876, 95% CI). Both techniques demonstrated outstanding specificity, exhibiting no cross-reactivity with bacteria from the same or closely related genera. A total of 74 fish samples suspected to be infected with the fish disease were gathered, with 26 and 23 samples testing positive for Aeromonas hydrophila via LAMP and qPCR, respectively. The concordance analysis for LAMP and qPCR methods generated a Kappa value of 0.909 (0.778–1.000, 95% CI), signifying a high degree of diagnostic consensus. This study highlights that the LAMP assay eliminates the thermal cycle temperature change process of qPCR, uses lysate to crudely extract bacterial genomic DNA, and can complete the detection within 40 min, rendering it a practical and efficient alternative for monitoring disease outbreaks at aquaculture sites. |
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| issn | 2218-1989 |
| language | English |
| last_indexed | 2024-03-11T00:50:48Z |
| publishDate | 2023-07-01 |
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| series | Metabolites |
| spelling | doaj.art-5a829dbb07fa4ee59c9b0891fb77d4e62023-11-18T20:27:59ZengMDPI AGMetabolites2218-19892023-07-0113784110.3390/metabo13070841Establishment of a Rapid LAMP Assay for <i>Aeromonas hydrophila</i> and Comparison with the Application of qPCRZihui Gao0Chunhua Yang1Xiaobo Zhang2Bing Hu3Huang Zhang4Zhihong Zhang5Wendong Kuang6Qiuyue Zheng7Jijuan Cao8Key Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, ChinaInstitute of Biological Resources, Jiangxi Academy of Sciences, Nanchang 330096, ChinaKey Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, ChinaKey Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, ChinaGuangzhou Double Helix Gene Technology Co., Ltd., Guangzhou 510320, ChinaInstitute of Biological Resources, Jiangxi Academy of Sciences, Nanchang 330096, ChinaInstitute of Biological Resources, Jiangxi Academy of Sciences, Nanchang 330096, ChinaKey Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, ChinaKey Laboratory of Biotechnology and Bioresources Utilization of Ministry of Education, College of Life Science, Dalian Minzu University, Dalian 116600, ChinaThe development of an exceptionally sensitive diagnostic technique for early identification of aquaculture diseases, specifically Aeromonas hydrophila, is essential for efficient management of disease outbreaks at aquaculture locations. In this research, a swift and sensitive diagnostic assay employing Loop-mediated isothermal amplification (LAMP) of Aeromonas hydrophila was devised and compared to the conventional qPCR method documented by Rong Wang. Validation of the diagnostic assay was carried out using actual samples obtained from aquaculture fish. The findings revealed that based on the rapid detection of crude bacterial genomic DNA, the fluorescent LAMP assay possessed a lower limit of detection (LOD) of 0.559 ng/μL (0.315–1.693, 95% CI), while the LOD for qPCR stood at 4.301 ng/μL (2.084–8.876, 95% CI). Both techniques demonstrated outstanding specificity, exhibiting no cross-reactivity with bacteria from the same or closely related genera. A total of 74 fish samples suspected to be infected with the fish disease were gathered, with 26 and 23 samples testing positive for Aeromonas hydrophila via LAMP and qPCR, respectively. The concordance analysis for LAMP and qPCR methods generated a Kappa value of 0.909 (0.778–1.000, 95% CI), signifying a high degree of diagnostic consensus. This study highlights that the LAMP assay eliminates the thermal cycle temperature change process of qPCR, uses lysate to crudely extract bacterial genomic DNA, and can complete the detection within 40 min, rendering it a practical and efficient alternative for monitoring disease outbreaks at aquaculture sites.https://www.mdpi.com/2218-1989/13/7/841<i>Aeromonas hydrophila</i>LAMPqPCR |
| spellingShingle | Zihui Gao Chunhua Yang Xiaobo Zhang Bing Hu Huang Zhang Zhihong Zhang Wendong Kuang Qiuyue Zheng Jijuan Cao Establishment of a Rapid LAMP Assay for <i>Aeromonas hydrophila</i> and Comparison with the Application of qPCR Metabolites <i>Aeromonas hydrophila</i> LAMP qPCR |
| title | Establishment of a Rapid LAMP Assay for <i>Aeromonas hydrophila</i> and Comparison with the Application of qPCR |
| title_full | Establishment of a Rapid LAMP Assay for <i>Aeromonas hydrophila</i> and Comparison with the Application of qPCR |
| title_fullStr | Establishment of a Rapid LAMP Assay for <i>Aeromonas hydrophila</i> and Comparison with the Application of qPCR |
| title_full_unstemmed | Establishment of a Rapid LAMP Assay for <i>Aeromonas hydrophila</i> and Comparison with the Application of qPCR |
| title_short | Establishment of a Rapid LAMP Assay for <i>Aeromonas hydrophila</i> and Comparison with the Application of qPCR |
| title_sort | establishment of a rapid lamp assay for i aeromonas hydrophila i and comparison with the application of qpcr |
| topic | <i>Aeromonas hydrophila</i> LAMP qPCR |
| url | https://www.mdpi.com/2218-1989/13/7/841 |
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