Novel Peptide Nucleic Acid Melting Array for the Detection and Genotyping of Toxoplasma gondii
0
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
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.
Abstract
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.
참고문헌 (20)
- Nielsen PE, "Peptide nucleic acid (PNA). A DNA mimic with a peptide backbone", Bioconjug. Chem, 5, pp.3~7 (1994)
- Egholm M, "PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules", Nature, 365, pp.566~568 (1993)
- Stender H, "PNA for rapid microbiology", J. Microbiol. Methods, 48, pp.1~17 (2002)
- Lehtola MJ, "Fluorescence in situ hybridization using peptide nucleic acid probes for rapid detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in potable-water biofilms", Appl. Environ. Microbiol, 72, pp.848~853 (2006)
- Montoya JG, "Toxoplasmosis", Lancet (London, England), 363, pp.1965~1976 (2004)
- 채종일, "Laboratory passage and characterization of an isolate of Toxoplasma gondii from an ocular patient in Korea", Parasites, Hosts and Diseases, 41(3), pp.147~154 (2003), 대한기생충학ㆍ열대의학회
- Kim WJ, "Peptide nucleic acid based molecular authentication for identification of four medicinal Paeonia species using melting array analysis of the internal transcribed spacer 2 region", Molecules, 22 (2017)
- Gupta P, "Single nucleotide polymorphisms:a new paradigm for molecular marker technology and DNA polymorphism detection with emphasis on their use in plants", Curr. Sci, 80, pp.524~535 (2001)
- Dubey J, "Advances in the life cycle of Toxoplasma gondii", Int. J. Parasitol, 28, pp.1019~1024 (1998)
- 송경주, "Seroprevalence of toxoplasmosis in Korean pregnant women", Parasites, Hosts and Diseases, 43(2), pp.1~10 (2005), 대한기생충학ㆍ열대의학회
- 김능희, "서울지역 사육 소의 큐열 및 톡소포자충 항체 보유율 조사", 한국동물위생학회지, 38(4), pp.233~239 (2015), 한국동물위생학회
- Cheong KS, "A studies on toxoplasmosis antibody from slaughtered pigs and cattle", Korean J. Vet. Serv, 17, pp.32~36 (1994)
- Jones JL, "Foodborne toxoplasmosis", Clin. Infect. Dis, 55, pp.845~851 (2012)
- Racka K, "Survey of Toxoplasma gondii antibodies in meat juice of wild boar (Sus scrofa) in several districts of the Czech Republic", Ann. Agric. Environ. Med, 22, pp.231~235 (2015)
- Howe DK, "Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis", J. Clin. Microbiol, 35, pp.1411~1414 (1997)
- Ajzenberg D, "Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings", J. Infect. Dis, 186, pp.684~689 (2002)
- Khan A, "Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains", J. Clin. Microbiol, 43, pp.5881~5887 (2005)
- Sibley LD, "Virulent strains of Toxoplasma gondii comprise a single clonal lineage", Nature, 359, pp.82~85 (1992)
- Mordue DG, "Acute toxoplasmosis leads to lethal overproduction of Th1cytokines", J. Immunol, 167, pp.4574~4584 (2001)
- Komiyama M, "PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection", J. Am. Chem. Soc, 125, pp.3758~3762 (2003)