title: Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.
abstract: Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6.
authors: Department of Microbiology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea; Department of Biomedical Science, Graduate School of Ajou University, Suwon 443-721, Republic of Korea.
authors: Heekyoung
authors: H
authors: Kang
authors: Department of Microbiology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea; Department of Biomedical Science, Graduate School of Ajou University, Suwon 443-721, Republic of Korea.
authors: Gi-Sang
authors: GS
authors: Seong
authors: Department of Microbiology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea; Department of Biomedical Science, Graduate School of Ajou University, Suwon 443-721, Republic of Korea.
authors: Hae-Jin
authors: HJ
authors: Sohn
authors: Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors: Jong-Hyun
authors: JH
authors: Kim
authors: Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea.
authors: Sang-Eun
authors: SE
authors: Lee
authors: Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea.
authors: Mi Yeoun
authors: MY
authors: Park
authors: Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea.
authors: Won-Ja
authors: WJ
authors: Lee
authors: Department of Microbiology, Ajou University School of Medicine, Suwon 443-721, Republic of Korea; Department of Biomedical Science, Graduate School of Ajou University, Suwon 443-721, Republic of Korea. Electronic address: hjshin@ajou.ac.kr.
authors: Ho-Joon
authors: HJ
authors: Shin
mesh_terms: D000562:Amebiasis / Q000175:diagnosis*; D000818:Animals; D020808:Central Nervous System Protozoal Infections / Q000175:diagnosis*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D051379:Mice; D016848:Naegleria fowleri / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000592:standards; D015203:Reproducibility of Results
chemical_list: D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer
keywords: ITS primer; Nae3 primer; Naegleria fowleri; Nfa1 primer; PAM