Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification assay for detection of Strongyloides stercoralis
10.16250/j.32.1915.2025220
- VernacularTitle:基于荧光重组酶介导的等温扩增技术检测 粪类圆线虫方法的建立及初步评价
- Author:
Xiaodan CHEN
1
;
Wanqiong CHENG
1
;
Xiaoyin FU
1
;
Jiayin LÜ
2
;
Jiayue SUN
3
;
Qiuhua BAI
1
;
Xue HAN
4
;
Yunliang SHI
5
;
Dengyu LIU
1
Author Information
1. School of Basic Medical Sciences, Guangxi Medical University, Nanning, Guangxi 530021, China; Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Guangxi Zhuang Autonomous Region Department of Education, Nanning, Guangxi 530021, China
2. Department of Clinical Laboratory, Women’s Hospital, School of Medicine, Zhejiang University, China
3. Department of Clinical Laboratory, Dapeng Branch, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital, China
4. The Fifth Affiliated Hospital of Guangxi Medical University, China
5. School of Basic Medical Sciences, Guangxi Medical University, Nanning, Guangxi 530021, China; Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Guangxi Zhuang Autonomous Region Department of Education, Nanning, Guangxi 530021, China; Guangxi Higher Education Engineering Research Center of Advanced Technology for Medical Bio-manufacturing, Nanning, Guangxi 530021, China
- Publication Type:Journal Article
- Keywords:
Strongyloides stercoralis;
Fluorescent recombinase-aided amplification;
Nucleic acid detection;
18S rRNA gene;
Rapid detection
- From:
Chinese Journal of Schistosomiasis Control
2026;38(2):160-168
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a fluorescent recombinase-aided amplification (RAA) assay for detection of Strongyloides stercoralis nucleic acid and to preliminarily evaluate its performance. Methods Six sets of specific primers targeting S. stercoralis 18S ribosomal RNA (18S rRNA) gene and one fluorescent probe were designed and synthesized. The optimal primer-probe set was determined through systematic screening and optimization to establish the fluorescent RAA assay. The assay was evaluated using S. stercoralis genomic DNA at concentrations of 100, 10, and 1 pg/μL, and 100, 10, and 1 fg/μL, as well as recombinant pUC57 plasmids containing the target gene fragments at 1 × 105, 1 × 104, 1 × 103, 1 × 102, 1 × 101, 1 × 100 copies/reaction, to determine the analytical sensitivity. Genomic DNA from Ascaris lumbricoides, Ancylostoma duodenale, Enterobius vermicularis, Angiostrongylus cantonensis, Trichinella spiralis, Clonorchis sinensis, Schistosoma japonicum, and Taenia saginata was used to assess assay specificity. A total of 25 stool samples from patients suspected of S. stercoralis infection were tested by the modified Baermann funnel technique, PCR, and the established fluorescent RAA assay. The sensitivity, specificity, concordance rate and their 95% confidence intervals (CI) of these three techniques were estimated, and agreement between methods was evaluated using the Kappa coefficient. Results Exo-4 was identified as the optimal primer set screened from the six primer sets, and the best amplification performance was achieved when the final concentrations of the forward and reverse primers were 0.44 μmol/L and a probe concentration was 0.20 μmol/L. The limit of detection of the fluorescent RAA assay was 100 fg/μL for genomic DNA of S. stercoralis and 1 × 100 copies/reaction for recombinant plasmids. Specific fluorescence signals were detected within 5 min, with no cross-reactivity observed with A. lumbricoides, A. duodenale, E. vermicularis, A. cantonensis, T. spiralis, C. sinensis, S. japonicum, or T. saginata. Among the 25 clinical stool samples from patients suspected of S. stercoralis infections, the modified Baermann funnel technique and fluorescent RAA assay detected 19 positives and 6 negatives, whereas PCR detected 18 positives and 7 negatives. The fluorescent RAA assay showed a sensitivity of 100.00% [95% CI: (82.35%, 100.00%)], specificity of 100.00% [95% CI: (54.07%, 100.00%)], concordance rate of 100.00% [95% CI: (86.28%, 100.00%)], and a Kappa coefficient of 1.00 [95% CI: (1.00, 1.00)] (P < 0.001) relative to the modified Baermann funnel technique, and a sensitivity of 100.00% [95% CI: (81.47%, 100.00%)], specificity of 85.71% [95% CI: (42.13%, 99.64%)], concordance rate of 96.00% [95% CI: (79.65%, 99.90%)], and a Kappa coefficient of 0.90 [95% CI: (0.70, 1.00)] (P < 0.001). Positive amplification products emitted green fluorescence under a portable blue-light device, enabling visual interpretation of results. Conclusions The fluorescent RAA assay established in this study is rapid, highly sensitive, and highly specific. It enables detection of S. stercoralis nucleic acid under isothermal conditions and allows visual interpretation of results, providing a novel tool for rapid clinical diagnosis and field screening of S. stercoralis infections.
