Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

Autor: Kang H; 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., Seong GS; 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., Sohn HJ; 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., Kim JH; Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea., Lee SE; Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea., Park MY; Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea., Lee WJ; Division of Malaria and Parasitic Disease, Korea National Institute of Health, Osong 363-951, Republic of Korea., Shin HJ; 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.
Jazyk: angličtina
Zdroj: European journal of protistology [Eur J Protistol] 2015 Oct; Vol. 51 (5), pp. 401-8. Date of Electronic Publication: 2015 Aug 14.
DOI: 10.1016/j.ejop.2015.07.003
Abstrakt: 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.
(Copyright © 2015 Elsevier GmbH. All rights reserved.)
Databáze: MEDLINE
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