Jan 07, 2022
Detection of Cryptosporidium in stool sample by PCR-RFLP
- 1University of Ghana
- Ayi and Pawlowic Lab Collaborations
Protocol Citation: botchiesenyo 2022. Detection of Cryptosporidium in stool sample by PCR-RFLP. protocols.io https://dx.doi.org/10.17504/protocols.io.bxaxpifn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: August 11, 2021
Last Modified: January 07, 2022
Protocol Integer ID: 52279
Abstract
Nested PCR-RFLP adapted from Nichols, R.A.B., Campbell, B.M. and Smith, H.V., 2003. Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters and drinking waters by using a modified nested PCR-restriction fragment length polymorphism assay. Applied and environmental microbiology, 69(7), p.4183.
Materials
- GoTaq MasterMix from Promega
- Primers
Nested PCR
Nested PCR
12h 45m
12h 45m
Extract whole genomic DNA from stool samples.
Positive control DNA for Cryptosporidium parvum may be obtained from ATCC. Alternatively you can extract genomic DNA from wild type Cryptosporidium parvum purchased from Bunchgrass Farms (Deary, Idaho, USA), Waterborne Inc (New Orleans, Louisiana, USA), or Sterling Labs (Tucson, Arizona, USA).
3h
First round of PCR to amplify 18SrRNA
4h
Combine the following in a PCR tube:
5 μl of DNA (from positive control, experimental sample, or water for negative control)
0.1 μM Forward Primer ("out NDIAGF2": CAATTGGAGGGCAAGTCTGGTGCCAGC)
0.1 μM Reverse Primer ("out NDIAGR2": CCTTCCTATGTCTGGACCTGGTGAGT)
1x GoTaq Master Mix (Promega)
Ultrapure water to bring total volume to 25 microliters
30m
Thermocycler program:
95 ºC -5 minutes,
30 cycles:
94 ºC -30 seconds
58 ºC - 1 minute
72 ºC - 30 seconds
3h 30m
Second round of PCR to amplify 18SrRNA
4h 30m
Combine the following in a PCR tube:
1 μl of PCR product from Step 2
0.1 μM Forward Primer ("DIAGF": AAGCTCGTAGTTGGATTTCTG)
0.1 μM Reverse Primer ("DIAGR": TAAGGTGCTGAAGGAGTAAGG)
1x GoTaq Master Mix (Promega)
Ultrapure water to bring total volume to 25 microliters
30m
Thermocycler program:
95 ºC -5 minutes,
45 cycles:
94 ºC -30 seconds
58 ºC - 1 minute
50 ºC - 1 minute
4h
Run 7 μl of PCR product from Step 3 on a 2% agarose gel and visualise by staining with ethidium bromide.
45m
RFLP
RFLP
4h 45m
4h 45m
Combine the following and incubate at 37 ºC for 4 hours:
5 μl PCR product from step 4 above
20u Restriction enzyme (Ssp1, Vsp1, Ase1)
1x final concentration Restriction enzyme digestion buffer (corresponds to enzyme used)
1x final concentration BSA
4h
Run digested PCR product from Step 5 on a 2% agarose gel and visualize by staining with ethidium bromide.
45m