Hello, I want to identify Cryptosporidium and Giardia from samples of rivers, so I wanted to know if it's better to use a nested PCR and what protocol should I use. Thanks.
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 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.
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
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