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

Peptide nucleic acid (PNA) probes, developed in the early 1990s, are DNA mimics, in which the negatively charged sugar-phosphate backbone is replaced by an achiral, neutral polyamide backbone formed by repetitive N-(2-aminoethyl) glycine units [1]. PNA can hybridize to complementary nucleic acid targets according to the Watson-Crick base-pairing rules [2]. Their unique chemical characteristics have been studied extensively owing to their research and diagnostic applications, e.g., in genomics, human pathology, virology, mycology, and bacteriology [3]. PNA molecules have unique hybridization characteristics; they exhibit rapid and strong binding to complementary targets and a lack of electrostatic repulsion [4]. Toxoplasma gondii, an opportunistic intracellular parasite, has the ability to infect any eukaryotic cell. It can infect all warm-blooded mammals, including humans [5]. Felids are the definitive hosts of T. gondii. Environmental contamination is commonly caused by the secretion of oocysts, which become infective upon sporulation, by felids. Intermediate hosts can be infected by T. gondii and produce tissue cysts in a number of organs, such as the skeletal muscle and central nervous system. When ingested in raw or uncooked food, these tissue cysts are Despite differences in virulence between strains of Toxoplasma gondii, rapid and accurate genotyping methods are lacking. In this study, a method was developed to detect and genotype T. gondii in food and environmental samples using PCR and a novel peptide nucleic acid (PNA) melting array. An alignment of genome sequences for T. gondii type I, II, and III obtained from NCBI was generated, and a single nucleotide polymorphism analysis was performed to identify targets for PCR amplification and a PNA melting array. Prior to the PNA melting array, conventional PCR was used to amplify GRA6 of T. gondii. After amplification, the PNA melting array was performed using two different PNA hybridization probes with fluorescent labels (FAM and HEX) and quenchers. Melting curves for each probe were used to determine genotypes and identify mutations. A 214-bp region of the GRA6 gene of T. gondii was successfully amplified by PCR. For all T. gondii strains (type I, II, and III) used to evaluate specificity, the correct genotypes were determined by the PNA melting array. Non-T. gondii strains, including 14 foodborne pathogens and 3 protozoan parasites, such as Giardia lamblia, Cryptosporidium parvum, and Entamoeba histolytica, showed no signal, suggesting that the assay has a high specificity. Although this is only a proof-of-concept study, the assay is promising for the fast and reliable genotyping of T. gondii from food and environmental samples.