Hello 

I advice you in trying PCR not Nested PCR and follow this link to see this nice work 

https://www.researchgate.net/profile/Hadeel_Albayati/publication/315824388_molecular_study_of_Cryptosporidium_spp_and_Giardia_lamblia_which_cause_diarrhea_in_camels_Camillus_dromedaries_in_Al-Diwaniyah_and_Al-Najaf_provinces_Iraq/links/590a05cda6fdcc4961774c78/molecular-study-of-Cryptosporidium-spp-and-Giardia-lamblia-which-cause-diarrhea-in-camels-Camillus-dromedaries-in-Al-Diwaniyah-and-Al-Najaf-provinces-Iraq.pdf?origin=publication_detail&ev=pub_int_prw_xdl&msrp=W1Gr6iSporiKlo_8O90C_L8zV-2rr16R7UNiDCdeVDzMrjtfZLpBO0bJiVOfK6voFUljm-5cf3OJye3Ytov7ZsB29Axf9VO6coajIJUmb7c.WrlMXLQqIK7KCuEzfrWa88kx5s-eZWK-uZ04H8cXxRocU1ZqWiuuOyp-JKKvXN5TLjc9yjC3KAJ5wIvMDcFQtA.IRhXLLsFgM_f1S0nS5WXTot5QQCJS8_mYIjPMP8MssVPuRQaXT2ezLX4dWg_G8P9EWBKgoqsBmSjoMh7-e3sww.uIPOaSgVHcpf8aqvJp_N8TxjB4nyzg5PPCsPVERqD9MKudgd3yFHVn3AgaTXNStuTjAtiD4OGwqZq9gA7Qz1Zw

Article molecular study of Cryptosporidium spp and Giardia lamblia w...

Hi colleague

Nested PCR is recommended in like your project.

hi dr. Nested PCR give  good result more than conventional PCR

Good luck  

Hi

Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide

range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust

nature of the oocyst’s walls, which necessitates more stringent treatments for disruption and recovery of DNA

for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts,

investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and

moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental

water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum

oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special

laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR

mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum

oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time

PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally

in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration

and concentration of spiked water samples. The addition of small amounts of BSA (5–20 ng/µl) to PCR and

TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and

above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from

spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of

the 18S rRNA gene.

I agree  with  Dr noor

Thank you all for your answers I will follow your advice 

PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally

in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration