Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR

Abstract

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.

FIG. 1.

Nested PCR for the Cpgp40/15 gene and the SSU rRNA. (A) Nested PCR with templates of four kinds of parasites. Nested PCRs for the SSU rRNA gene and the Cpgp40/15 gene are shown in lanes 1 to 4 and 5 to 8, respectively. Lanes 1 and 5, C. muris RN66 strain; lanes 2 and 6, C. parvum Iowa strain; lanes 3 and 7, G. lamblia H3 strain; lanes 4 and 8, G. muris Roberts-Thompson strain. (B) Nested PCR with templates of C. parvum and C. hominis strains. Nested PCRs for the Cpgp40/15 and SSU rRNA are shown in lanes 1 to 3 and 4 to 6, respectively. Lanes 1 and 4, C. parvum Iowa strain; lanes 2 and 5, C. parvum HNJ-1 strain; lanes 3 and 6, C. hominis Ogose strain. Lanes M and N show DNA size standards (100-bp DNA ladder; Takara Shuzo) and the PCR negative control, respectively.

FIG. 2.

RFLP of the Cpgp40/15 PCR product generated from three C. parvum and C. hominis strains. The products digested with HindIII (A), MflI (B), and RsaI (C) are in lanes 1 to 3, 4 to 6, and 7 to 9, respectively. Lanes 1, 4, and 7, C. parvum Iowa strain; lanes 2, 5, and 8, C. parvum HNJ-1 strain; lanes 3, 6, and 9, C. hominis Ogose strain. Lane M shows a 100-bp DNA ladder. The lower band of the Ogose strain digested with RsaI (lane 9) consists of two fragments of 160 and 170 bp.

FIG. 3.

Detectable limit of nested PCR. Nested PCR was performed with 10-fold serially diluted chromosomal DNA template of the C. parvum Iowa strain ranging from 5 × 10⁻⁹ g (lane 1) to 5 × 10⁻¹⁴ g (lane 6).

FIG. 4.

Relationship between genetic detection by nested PCR and oocyst number confirmed by FA from application experiments with oocyst-inoculated turbid water samples. Circles show successful detection by nested PCR. Solid circles show successful detection in all three reaction systems, and open circles show detection in at least one of the reaction systems. Crosses show failed detection by nested PCR.