Md. Abdul Khaleque
Department of Parasitology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Farjana Boby
Department of Parasitology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Md. Shahiduzzaman*
Department of Parasitology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Cryptosporidium, Human child, Animal, Bangladesh
Laboratory at the Department of Parasitology, Bangladesh Agricultural University, Mymensingh
Pest Management
Samples: Stool samples were collected from Shurjo Kanti (S.K.) Hospital, Mymensingh district of Bangladesh and fecal sample from cattle of Bangladesh Agricultural University, Mymensingh. The stool and fecal samples were initially processed at the collection sites and then brought to the Laboratory at the Department of Parasitology, Bangladesh Agricultural University, Mymensingh. Part of the work was done at the Laboratory of Department of Microbiology & Hygiene, and Department of Pathology, Bangladesh Agricultural University Mymensingh. The study was conducted during the period of January 2016 to June 2016. The samples were initially screened by by Ziehl-Neelsen technique and positive samples (9 samples from human and 1 sample from animal) were subjected to concentration of oocysts and DNA extraction.
DNA extraction Purelink™ Genomic DNA Mini Kit (Invitrogen, USA) was used to extract the DNA following manufacturer instructions.
Polymerase chain reaction (PCR) A primary PCR was performed by targeting the Small Sub Unit (SSU) rRNA gene of Cryptosporidium to confirm the detection of Cryptosporidium from stool samples according to the protocol described by Xiao et al., (1999) with some modifications. A PCR product of 1,325 bp from target gene was amplified by forward primer (5'-TTCTAGAGCTAATACATGCG-3) and reverse primer (5'-CCCATTTCCTTCG AAACAGGA -3'), (IDT, USA). PCR reaction was performed in a total 50 µl reaction volume containing PCR Master mix (Promega, USA) 45 µl, forward primer 1 µl, reverse primer 1 µl, deionized water 1 µl, and DNA template 2 µl. A total of 35 cycles were carried out, each consisting of 94°C for 3 min in case of initial denaturation, 94°C for 45 sec in case of denaturation, 54°C for 45s in case of annealing, 72°C for 1 min in case of extension and 72°C for 7 min in case of final extension, one kb DNA ladder (Promega, USA) was used to compare the band size of target DNA.
Agarose gel electrophoresis PCR products were analyzed in 1.5% agarose gel, stained with ethidium bromide and examined against UV light using an image documentation system. Genotyping For genotyping PCR products of different DNA samples were subjected to digestion by restriction enzyme. VspI (AseI) restriction enzyme (Lithuania, EU). For restriction fragment analysis, 10 µl of the PCR product was digested in a total of 25µl of reaction mixture, consisting of 1µl of VspI (AseI) restriction enzyme (Lithuania, EU), 5µl of respective restriction buffer and 9µl of distilled water at 37° C for 1 h, according to manufacturer’s instructions. The digested products were fractionated on 2.0% agarose gel and visualized by ethidium bromide staining.
2016 © International Journal of Applied Research 2(3) 122-126
Journal