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 x 10(6)), simple boiling with 10 mu l of PBS (pH 7.4) at 100 degrees C for 30 min was found to be the most rapid and efficient procedure, allowing amplification of 2.5 x 10(2) trophozoites using the Nfa-1 primer. The primers Nfal 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 Nfal primer was able to amplify the N. fowleri DNA 4 days post infection with 1 ng/mu l 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 Nfal primer at day 6. (C) 2015 Elsevier GmbH. All rights reserved.