Title : Simple PCR-NALFIA targeting SSU rRNA gene for sensitive detection of Vittaforma corneae in MKC patients
Abstract:
Background: Microsporidial keratoconjunctivitis (MKC) is an emerging ocular disease, with an increase in cases being recorded in tropical countries. Diagnosis is difficult due to non-specific characteristics and low pathogen load in a corneal scraping sample. Unlike Gram-chromotrope staining, the Nucleic Acid Lateral Flow Immunoassay (NALFIA) has much greater detection rates. Furthermore, since Thai isolates have significant genetic homogeneity, developing a standardized NALFIA technique becomes very cost-effective and appropriate for providing universal disease surveillance coverage across Thailand.
Objectives: To develop a rapid simple innovative approach: a species-specific PCR-NALFIA for detecting Vittaforma corneae targeting highly conserved SSU rRNA gene region
Methods: The study comprises the construction of a NALFIA paired with PCR targeting Vittaforma corneae highly conserved SSU rRNA gene using FITC-LPW20469 and BIOTIN-LPW20470 primers. The analytical sensitivity of the proposed test strip is being assessed using dilutional titers to determine the limit of detection (LoD).
Results: The PCR-NALFA test strip, utilizing the FITC-LPW20469 and BIOTIN-LPW20470 primers, was successfully developed and optimized for the detection of V. corneae amplicons. The first phase establishes the test strip's suitability for quick visual detection. Analytical validation through dilutional titrations to obtain the exact LoD.
Conclusion: The successful initial development of the PCR-NALFIA test strip offers a promising, user-friendly diagnostic platform. Leveraging the extreme genetic homogeneity and close relatedness of V. corneae isolates, this standardized test strip approach is expected to provide a highly sensitive, cost-effective, and rapid diagnostic tool for MKC. Once clinical validation is complete, it holds strong potential for both routine clinical use in Thailand and international disease surveillance.
