Development of a colorimetric loop-mediated isothermal amplification assay for detection of pathogenic Streptococcus species in aquaculture

Initially thought to be resistant to most bacterial infections, outbreaks in aquatic farms showed that tilapias are indeed susceptible to various diseases such as streptococcosis. Infections by Streptococcus spp. particularly Streptococcus agalactiae and Streptococcus iniae have been reported to cause major outbreaks affecting tilapia producers and farmers. Rapid and accurate detection of pathogens with a point-of-care (POC) application is compulsory to reduce the losses due to the disease. While the polymerase chain reaction (PCR) is a widely used conventional technique for DNA amplification in pathogen detection, it has some disadvantages such as slightly cost-inefficient, equipment complexity, necessitates precise temperature cycling and routine post-amplification analysis; and prone to false-positives results. Alternative technique such as the loop mediated isothermal amplification (LAMP) generally offers a simpler and more cost-effective alternative, specifically in terms of equipment requirements, simplicity of operation and reduced risk of false positives. These attributes make LAMP a promising choice for field-based testing and POC applications where rapid and reliable nucleic acid amplification is essential. Despite the advantages of LAMP in terms of simplicity, rapidity, and sensitivity, there is still a gap in the development of a specific LAMP assay tailored to accurately and reliably detect specific pathogens for enhanced disease management and surveillance. This project intends to address this gap by designing, optimising, and validating a novel and specific colorimetric detection tool using gold nanoparticles (AuNPs) probe conjugated with serotype-specific capsular polysaccharide subunit (cpsI) gene of S. agalactiae TP 486 E (serotype III) and species-specific superoxide dismutase (sodA) gene of S. iniae TSK-2 - via LAMP assay; that offers improved accuracy, and ease of use for the detection of S. agalactiae and S. iniae, thus aiding to the advancement of molecular diagnostics and its application in aquaculture sector. The LAMP reaction was initially optimised before the AuNPs probe preparation. The hybridisation of the LAMP amplicon and AuNP probe was then optimised for the best visualisation for assay detection. The specificity and sensitivity of the assay were tested to compare the efficiency of the developed assay with conventional PCR. LAMP amplicons were successfully amplified using the designated LAMP primers. The optimal LAMP reaction condition of the cpsI gene in TP 486 E comprises 6 mM MgSO4 with the incubation temperature and time of 65ºC for 60 min, respectively. Meanwhile, the optimal LAMP reaction condition of the sodA gene in TSK-2 was 6 mM MgSO4 with incubation temperature and time of 65ºC for 45 min, respectively. The optimal ratio and most distinct colour for visualisation of the assay after salt induction for both strains was at 4:6 of LAMP amplicon: AuNP probe. On the other hand, 0.05 mM of MgSO4 was selected as the best salt concentration for aggregation for both strains. No cross-reactivity was detected in the LAMP assay when tested along the other non-specific aquatic pathogens indicating a high-specificity reaction. The detection limit of bacterial cells for TP 486 E was 4×103 CFU/ml, which is 100 times more sensitive than the conventional PCR assay (4×105 CFU/ml). The sensitivity of TSK-2 using the LAMP-AuNP assay was 3.3×103 CFU/ml, which was 10 times higher than the PCR assay (3.3×104 CFU/ml). Both the LAMPAuNP assay successfully detected the presence of S. agalactiae serotype III and S. iniae in the infected tilapia juveniles used in the bacterial challenge within approximately 1 hour. The diagnostic sensitivity, specificity and accuracy values for the LAMP-AuNP assay to detect S. agalactiae strain TP 486 E compared to the conventional PCR were 100.0, 93.75 and 98.33% respectively. On the other hand, the LAMP-AuNP method for the detection of S. iniae strain TSK-2 yielded 100% diagnostic sensitivity, specificity and accuracy in comparison to PCR assay. There was an excellent overall agreement between the LAMP-AuNP assay and conventional PCR for S. agalactiae strain TP 486 E (κ=0.957) and S. iniae strain TSK-2 (κ=1.0). The colorimetric detection by LAMP is comparatively similar to the PCR assay but has a shorter turnaround time from extraction to result detection. The developed LAMP-AuNP assay has great potential for use in the detection of pathogenic aquatic Streptococcus at the POC and limited resources and settings.

Text 122791.pdf Download (1MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18665

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