Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment

Abstract

A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites.

FIG. 1.

ITS PCR products generated from the DNA of various free-living amoebae using conserved primers only (c), conserved and species-specific primers (m), and species-specific primers only (s). (A) PCR results for three thermophilic Naegleria species. The N. fowleri long-ITS variant (fow L) and short-ITS variant (fow S) gave single bands of 453 and 409 bp, respectively, with the conserved primers and single bands of 311 bp with only the species-specific primers. With the multiplex PCR, the N. fowleri long-ITS variant displayed three bands of 453, 388 + 376, and 311 bp, whereas the N. fowleri short-ITS variant displayed four bands of 409, 376, 344, and 311 bp. N. australiensis (aust) and N. lovaniensis (lova) gave single fragments of approximately 400 bp with the conserved and the multiplex primers. (B) No PCR fragment was observed with Nuclearia spp. (Nuc) or Acanthamoeba spp. (Aca). Conserved and multiplex PCR gave single fragments of approximately 800, 600, and 500 bp with Hartmannella spp. (Har), Vahlkampfia spp. (Vah), and with Willaertia spp. (Wil), respectively. M, molecular markers (100-bp DNA ladder with a prominent 500-bp fragment).

FIG. 2.

Multiplex PCR analysis for the simultaneous detection of the human-pathogenic N. fowleri and the non-human-pathogenic isolates from the Dampierre site. Two banding patterns are observed in the gel. The pattern with three bands indicated the presence of the N. fowleri long-ITS variant (fow L). The single-band pattern corresponded to all other thermophilic Naegleria strains. N, negative control amplification in the absence of DNA; M, 100-bp DNA ladder with a prominent 500-bp fragment.

FIG. 3.

Geographical distribution of N. fowleri variants in France. The short-ITS variant was found only at Bugey and Golfech, and the long-ITS variant was found at the other sites analyzed. St Laurent, Cattenom, and Chooz were not examined in this study. Previous RAPD and ITS analyses showed that Cattenom and Chooz were colonized by variants closely related to the South Pacific variant. In addition, Cattenom was the only French site at which different variants were found.

FIG. 4.

MseI and NlaIV digestion patterns of the ITS PCR products for the long-ITS variant of N. fowleri (lane 1), the short-ITS variant of N. fowleri (lane 2), N. lovaniensis (lane 3), and N. australiensis (lane 4). M, 100-bp DNA ladder. On the right are the restriction maps of MseI and NlaIV for the long and the short N. fowleri variants (fow L and fow S), N. lovaniensis (lova), and N. australiensis (aust). The conserved sites are marked with asterisks.