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Development and application of a dual LAMP-LFD assay for the simultaneous detection of Streptococcus suis and
Haojie Wang1,2, Chenhui Dong2, Xiaoxiao Tian1
1State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
Introduction:
Streptococcus suis (S. suis) and Glaesserella parasuis (G. parasuis) are prevalent pathogens in pig populations and are often associated with co-infections, leading to substantial economic losses in the swine industry. However, there is currently a shortage of rapid detection methods. In this study, a dual loop-mediated isothermal amplification combined with lateral flow dipstick (LAMP-LFD) assay was developed for the simultaneous and convenient detection of S. suis and G. parasuis.
Methods:
The assay utilized primers targeting the conserved regions of the gdh gene of S. suis and the infB gene of G. parasuis. Optimal primer sets were identified, and reaction conditions, including temperature, time, and primer concentration ratios, were optimized using single-variable control method. The LAMP-LFD assay was established with biotin and digoxin or biotin and 6-FAM-labeled FIP/BIP primers, combined with LFD.
Results:
The assay was most effective at a reaction temperature of 62°C, a primer concentration ratio of 1:4, and a reaction time of 40 minutes. The minimum detection limits were 22 and 18 copies/μL for recombinant plasmids and 19 and 20 CFU for bacterial samples of S. suis and G. parasuis, respectively. The assay showed no cross-reactivity with other pathogens and exhibited high adaptability across various thermal platforms, including PCR instruments, metal baths, and water baths. Clinical testing of 106 samples revealed positive rates of 11.32% (12/106) for S. suis, 25.47% (27/106) for G. parasuis, and 2.83% (3/106) for mixed infections.
Discussion:
This simple, rapid, specific, and sensitive dual LAMP-LFD assay provides robust technical support for the prevention and control of swine streptococcosis and Glässer's disease.
