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| Visual Detection of Streptococcus iniae Based on Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick |
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Abstract Streptococcus iniae is a species belonging to Gram-positive coccus and produces beta hemolysis, which can infect a broad range of freshwater and marine fish species and lead to seriously economic losses in the aquaculture industry worldwide. Thus, a diagnostic method for rapid and accurate detection of S. iniae was urgently needed to effectively control the spread of streptococcicosis caused by S. iniae. In the present study, a LAMP-LFD method was developed to rapid detection of S. iniae, based on the loop-mediated isothermal amplification (LAMP) and visualization by a lateral flow dipstick (LFD). Three pairs of primers, i.e., gyrB-F3, gyrB-B3, gyrB-FIP, gyrB-BIP, gyrB-LF and gyrB-LB, were designed to recognize the gyrase subunit B (gyrB) gene of S. iniae; and among which the forward inner primer (gyrB-FIP) was biotinylated. A DNA probe (gyrB-HP) labeled by fluorescein isothiocyanate (FITC) was also designed specifically to recognize the gyrB gene. The biotinylated LAMP was performed and the LAMP product was hybridized with FITC-labeled probe and detected by LFD. The optimized assay could detect S. iniae by incubation at 65°C for 30 min, and under such conditions, the initiation time for positive amplification gradually increased as the concentration of template decreased, showing a typical linear relationship. Starting from isothermal amplification, the whole detection procedure needed only 40 min, which reduced by 2 h compared to conventional PCR assay. The LAMP-LFD could specifically detect S. iniae isolate and no amplification was observed from other pathogenic bacteria, e.g., the Streptococcus (i.e., Streptococcus dysgalactiae subsp., and et al), multiple Gram-positive bacteria (i.e., Staphylococcus aureus, Nocardia seriolae, and et al), and multiple Gram-negative bacteria (i.e., Vibrio harveyi, Pseudomonas aeruginosa, and et al). The detection limit of LAMP-LFD for pure culture of S. iniae was 8.70×101 cfu/mL (1.74 cfu/reaction), which was 10 fold higher than LAMP and 100 times higher than the conventional PCR assay. It was all positively detected when taking pure gDNA of S. iniae of 8.70×101 cfu/mL (the detection limit) as template in triplicate, which indicated good reproducibility. In the case of artificially contaminated liver tissue of Lateolabrax japonicus, the detection limit was 4.35×103 cfu/mL, which was 100 times higher than the conventional PCR method as well. Infection by S. iniae was successfully detected from field samples by LAMP-LFD, which was quite coincident with the results obtained by conventional culture methods. Therefore, LAMP-LFD was an accurate, time-saving and easy-to-use method for detection of S. iniae, and can be used to on-site screening of S. iniae.
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Received: 17 April 2014
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