Exploration of fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for detection of Isospora suis oocysts
| Autor: | Meizhen Chen, Wei Zhou, Shoukun Wang, Cui-Qin Huang, Lingying Hu, Fuli Wen, Renfeng Chen, Liangping Yue |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Swine 030106 microbiology Immunology Loop-mediated isothermal amplification Biology Real-Time Polymerase Chain Reaction Sensitivity and Specificity Microbiology law.invention 03 medical and health sciences Feces Plasmid law RNA Ribosomal 18S Parasite hosting Animals DNA Primers Detection limit Electrophoresis Agar Gel Swine Diseases Isospora Oocysts General Medicine Nucleic acid amplification technique DNA Protozoan Isosporiasis biology.organism_classification Virology 030104 developmental biology Infectious Diseases Real-time polymerase chain reaction Microscopy Fluorescence Recombinant DNA Parasitology Nucleic Acid Amplification Techniques |
| Zdroj: | Experimental parasitology. 165 |
| ISSN: | 1090-2449 |
| Popis: | Isospora suis is an intestinal protozoan parasite in pigs. The 2-3 weeks old piglets are most often infected by I. suis because their immune system is not fully developed. The infection exhibits clinical features such as diarrhea and dehydration and seriously affects the economic interests of farmers. The traditional method of identifying I. suis relies on the detection of fecal oocysts, which depends heavily on the accumulation of experience. Thus, missed detection, and false alarms often occur during detection. With the development of molecular-based detection methods, development of a simple, convenient and more sensitive method for the detection of I. suis is an urgent need. In this study, based on the 18S rRNA gene sequence, a fluorescence -based real-time loop-mediated isothermal amplification (LAMP) assay was established for the detection of I. suis. The results showed that the assay is highly specific and sensitive, with a detection limit of 2.74 × 10(2) copies/μL recombinant plasmid of I. suis, corresponding to 1 fg/μL plasmid when converted to DNA concentration. The sensitivity is about 100 times higher than conventional PCR. Additionally, DNA extracted from a certain number of oocysts was used for detection, and it showed that the LAMP assay had a detection limit of 5 oocysts, lower than that of 13 oocysts of conventional PCR. The established LAMP assay overcomes the shortage of the traditional microscopy-based method, and provides a valuable way for molecular detection of I. suis. |
| Databáze: | OpenAIRE |
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