FIG. 5.

Multiplex PCR analysis for the detection of C. cayetanensis, C. parvum, and microsporidia in a stool sample with FTA filters. Two 100-μl composite stool samples obtained from a Nepali clinic were examined; one untreated sample and one sample artificially contaminated with 500 E. intestinalis spores were prepared and applied to FTA filters as described in Materials and Methods. The filters were then used as the initial template in a two-step multiplex PCR. Primer pairs F1E and R2B, CP-DIAGF and CP-DIAGR, and Micro-F and Micro-R were used during the first amplification. From 1 to 5 μl of the resulting product was then used in a second reaction with the primer pair F3E and R4B and microsporidia species-specific primers was then carried out (see Materials and Methods). Thermal cycling parameters for both sets of reactions were identical to those used for amplifying C. cayetanensis DNA.