Development of Novel Peptide Nucleic Acid Melting Array for the Detection and Genotyping of Toxoplasma gondii

Introduction: Although reasonable numbers of PCR and real-time PCR assays have been developed to identify T. gondii in animals and their meat products, the methods for genotyping T. gondii strains (type I, II and III) are limited to serological tests. Thus, there is a need for fast and reliable sequence-based assay for differentiating most virulent type I strain from less virulent type II and III strains.

Purpose: The purpose of this study was to detect T. gondii in food and environmental samples and differentiate genotypes using novel peptide nucleic acid (PNA) melting array.

Methods: Genomic information of T. gondii type I, II and III obtained from NCBI was aligned and SNP (single nucleotide polymorphism) analysis was performed to identify targets of PCR amplification and PNA melting array. Prior to the PNA melting array, conventional PCR amplified the GRA6 gene of T. gondii. After the amplification, PNA melting array was carried out using two different PNA hybridization probes labeled with fluorescent dyes (FAM and HEX) and quencher. Then melting temperature of each probe was analyzed to finalize genotype and possible mutation.

Results: Conventional PCR confirmed amplification of 214-bp region of GRA6 gene in T. gondii. Total of 8 T. gondii strains (3 type I, 3 type II and 2 type III) tested for the specificity showed exact genotypes with PNA melting array. Non-T. gondii strains including 14 different food-borne pathogens as well as 3 protozoan parasites such as Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica showed no amplification for the prior PCR, suggesting high specificity of the assay.

Significance: Although this is only a proof-of-concept study, the assay showed promising results for fast and reliable genotyping of T. gondii isolated from food and environmental samples.