Table 2.
Comparison of the used PCR approaches.
| Method | Sporocysts Isolation |
gDNA Extraction |
Number of Water Samples (n) |
Positive Samples * |
Cases | Species Detected **** |
|
|---|---|---|---|---|---|---|---|
| Positive ** | False Positive *** |
||||||
| Direct PCR (species-specific primers) |
Sucrose gradient |
“GeneJET Genomic DNA Purification Kit” |
20 | 0 (0.0%) | 0 | 0 | 0 |
| Nested PCR (species-specific primers only in 2nd round of PCR) |
Sucrose gradient |
“GeneJET Genomic DNA Purification Kit” |
35 | 8 (22.9%) | 12 | 2 | 2 |
| Sedimentation | “Genomic DNA Purification Kit” |
35 | 3 (8.6%) | 4 | 1 | 2 | |
| Filtration | “GeneJET Genomic DNA Purification Kit” |
35 | 14 (40.0%) | 15 | 1 | 3 | |
| Multiplex-nested PCR (species-specific primers only in 2nd round of PCR) |
Filtration | “GeneJET Genomic DNA Purification Kit” |
35 | 7 (20.0%) | 10 | 3 | 2 |
| Semi-nested PCR (species-specific primers) |
Filtration | “GeneJET Genomic DNA Purification Kit” |
35 | 0 (0.0%) | 0 | 0 | 0 |
| Nested PCR (species-specific primers in both rounds) |
Filtration | “GeneJET Genomic DNA Purification Kit” |
35 | 34 (97.1%) | 84 | 0 | 8 |
* The number of samples in which at least one of targeted Sarcocystis species was identified. ** The number of positive PCR fragments. *** Based on sequencing the number of incorrectly identified species. **** Based on sequencing the number of targeted species identified in samples.