Performing real-time PCR amplification

Four different specific primers targeting ribosomal genes of Cryptosporidium spp., Blastocystis sp., E. bieneusi, and Encephalitozoon spp., were selected (Table ​(Table88).

Primers used in this study.

EbITS-89F

EbITS-191R

TGTGTAGGCGTGAGAGTGTATCTG

CATCCAACCATCACGTACCAATC

Internal transcribed

spacer (ITS)

MSP1F

Eint227R

CACCAGGTTGATTCTGCCTGAC

CTAGTTAGGCCATTACCCTAACTACCA

JVAF

JVAR

ATGACGGGTAACGGGGAAT

CCAATTACAAAACCAAAAAGTCC

BHRMF

BHRMR

CGAATGGCTCATTATATCAGTT

AAGCTGATAGGGCAGAAACT

*The fragment size is different regarding the species.

Real-time PCR was carried out using Rotor-Gene Q (QIAGEN, Germany) real-time instrument. The real-time PCR reactions were conducted in a 15-μL total volume containing 7.5 μL of 2 × real-time PCR master mix (BIOFACT, Korea), 0.5 μL of each primer (5 ρM), 3.5 μL of distilled water, and 3 μL of template DNA. Amplification reactions were done as follows: 95 °C for 10 min followed by 40 cycles: 95 °C for 25 s, 59 °C for 30 s, 72 °C for 20 s, and ramping from 70 °C to 95 °C at 1°Cs⁻¹. Appropriate positive sequenced controls for each parasite together with sterile distillated water as negative controls were tested in each run. The real-time PCR assays were carried out in duplicate to check the reproducibility. The melting profiles were also analyzed using Rotor-Gene Q software to exclude non-specific amplifications and primer-dimers.

Real-time PCR results were considered negative when the Ct value was more than 38 or no amplification curve was obtained. All samples with Ct value above 35 were either retested or their melting curve were justified by the positive control to confirm the result.