Molecular and microscopic techniques for detection of Sarcocystis neurona sporocysts in fecal samples

ABSTRACT

Diagnosis of Sarcocystis sp. in the definitive host is generally by microscopic detection of the sporocysts in feces. This method is insensitive and cannot differentiate between species because sporocysts lack specific staining criteria. The hypothesis suggested that molecular techniques provide better alternatives to classical detection of Sarcocystis sporocysts. The sensitivity of two PCR assays was compared to one another and to microscopic examination by conventional fecal flotation and Diamant-Fuchsin staining procedures for detection of sporocysts spiked into mice feces. PCR1 assay using LSM1 and LSM2 primers that amplified 496 bp of the ssurRNA gene was more sensitive than the PCR2 method using JNB25 and JD396 primers that amplified 334 bp of a RAPD-derived marker. PCR1 gave positive results with 200 micro1 of fecal suspension spiked with as little as 5 sporocysts compared to 75 sporocysts detected by JNB25 and JD396 primers. PCR1 was more sensitive than conventional microscopy. PCR1 or PCR2 followed by sequencing or RFLP analysis not only detected Sarcocystis sporocysts in feces but also enabled to ascertain the genotype of the species as S. neurona