Identification of Balamuthia mandrillaris by PCR assay using the mitochondrial 16S rRNA gene as a target

Abstract

Balamuthia mandrillaris is an opportunistic pathogen that causes granulomatous amebic meningoencephalitis in animals, including humans. Based on sequence analysis of mitochondrial small-subunit-rRNA genes, we developed primers that amplify a Balamuthia-specific PCR product. These primers will be useful for retrospective analyses of fixed tissues and possible identification of Balamuthia in vivo.

FIG. 1.

B. mandrillaris multiplex rns PCR. Amplification was performed in 50-μl reaction mixtures with 2.5 mM MgCl₂; cycle conditions were 40 cycles of 1 min at 94°C, 2 min at 48°C, and 3 min at 72°C followed by a 15-min final extension at 72°C. Lanes 1, 6, and 10, 1-kb marker; lane 2, Balamuthia DNA with Balamuthia-specific primers; lane 3, Balamuthia DNA with Acanthamoeba-specific primers; lane 4, Acanthamoeba DNA with Balamuthia-specific primers; lane 5, Acanthamoeba DNA with Acanthamoeba-specific primers; lanes 7 to 9, Acanthamoeba and Balamuthia DNA with Balamuthia-specific primers (lane 7), with an Acanthamoeba-specific primer set (lane 8), and with both genus-specific primers sets (lane 9); lane 11, human DNA with both specific primer sets, lane 12, negative control. Amplicons produced by these mitochondrial 16S genus-specific primer sets are indicated on the right.