Specific recognition and detection of Singapore grouper iridovirus by an activatable aptamer LYGV1c

【Objective】 To construct an activated aptamer probe capable of specifically recognizing the Singapore grouper iridovirus（SGIV）, which could provide technical support for the development of rapid detection kits specifically recognizing SGIV, and achieve early diagnosis and precise prevention and control of SGIV infection. 【Method】 Based on aptamer LYGV1 which specifically recognizes SGIV-infected host cells, a target-activatable aptamer TAA-LYGV1c was successfully constructed through functional modifications, including the incorporation of T-strand, C-strand, fluorophore, and quencher groups. Subsequently, the specific recognition ability of TAA-LYGV1c for SGIV was systematically validated using flow cytometry, confocal laser scanning microscopy, and real-time fluorescence quantitative PCR. Optimal working concentration, incubation time, incubation temperature and detection limit for target cells were optimized to evaluate the practicality of TAA-LYGV1c. Finally, an SGIV infection model was established to further evaluate the practical application potential of TAA-LYGV1c in aquaculture production through comprehensive analysis using both flow cytometry and real-time fluorescence quantitative PCR. 【Result】 After the addition of TAA-LYGV1c, the fluorescence value of spleen（GS） cells in grouper in the SGIV infection group was extremely significantly higher than that in the SGIV infection group without the addition of TAA-LYGV1c（P＜0.01, the same below）, and was also extremely significantly higher than that in the blank control group and the nerve necrosis virus（NNV） infection group. It was indicated that TAALYGV1c could specifically recognize SGIV infection. The optimal working concentration of the TAA-LYGV1c was 500 nmol/L, the optimal incubation temperature was 28 ℃, and fluorescence could be detected at the shortest incubation time of 1 min. The minimum detection limits of different detection methods varied. Among them, the detection limit for target cell recognition by TAA-LYGV1c analyzed by flow cytometry was 5×10³ Cells/mL, while the minimum detection limits analyzed by fluorescence microplate reader and real-time fluorescence quantitative PCR were both 500 Cells/mL. That was, there was a certain connection between the detection sensitivity and the detection principle of the instrument. The flow cytometry analysis results based on TAA-LYGV1c were consistent with the real-time fluorescence quantitative PCR detection results, further confirming that the TAA-LYGV1c could specifically recognize SGIV infection, and also indicating that nucleic acid aptamers had potential application value in the detection of aquaculture pathogens. 【Conclusion】 The developed activatable aptamer TAA-LYGV1c has advantages including operational simplicity, high sensitivity and strong specificity. This probe enables rapid early diagnosis of SGIV infection and provides technical support for developing rapid detection kits specifically targeting SGIV.