Computational protocol: Molecular investigation of Cryptosporidium and Giardia in pre- and post-weaned calves in Hubei Province, China

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Protocol publication

[…] A total of 339 fresh faecal samples were collected (September to December 2016) from pre- and post-weaned calves (1 to 12 weeks old) from one beef farm (i.e. Suizhou) and five dairy farms (i.e. Chezhan, Guangming, Meijiadun, Qiaoner and Yangzijiang) in the north, east and west regions of Hubei Province, China (Fig. ). Faecal samples were collected rectally from individual calves and kept at 4 °C following sampling, and then frozen at −20 °C for subsequent DNA isolation and molecular testing. Genomic DNA was extracted from 0.2 g of each faecal sample using the PowerSoil DNA Isolation Kit (MoBio, Carlsbad, USA), according to the manufacturer’s protocol, and stored at −20 °C. This kit was used, as it is highly effective at removing components that are inhibitory to PCR [–]. Aliquots (2 μl) of individual genomic DNA samples were subjected to nested PCR-based amplification and sequencing, employing (individually) two distinct loci of nuclear ribosomal DNA in separate assays. For detection of Cryptosporidium, a portion of the large subunit of the nuclear ribosomal RNA gene (pLSUc; ~500 bp) was used [], and further genotypic/subgenotypic classification was achieved by employing a portion of the small subunit of the nuclear ribosomal RNA gene (pSSUc; ~ 590 bp) []. For the specific and assemblage-based classification of Giardia, a portion of the SSU gene (pSSUg; ~ 290 bp) was employed [, ] (Additional file : Table S1).Fig. 1 In brief, nested PCRs were carried out in 50 μl using a standard reaction buffer, 2.0–3.0 mM of MgCl2 (depending on the locus), 200 μM of each dNTP, 50 pmol of each primer and 1 U of Taq polymerase (Mango DNA polymerase, Bioline, London, UK) using established cycling protocols (Additional file : Table S1). Except for the no-template controls, 2 μl of genomic DNA were added to the primary PCR, from which 1 μl was carried over to the secondary PCR. Known test-positive, test-negative and no-template controls were included in each PCR run.All nested PCR products were detected by electrophoresis in ethidium bromide-stained (1.5%) agarose gels before sequencing. For sequencing, aliquots (5 μl) of individual amplicons (undigested) were treated with the enzymes Exo I and a thermosensitive alkaline phosphatase (FastAP, Thermofisher, Carlsbad, USA), according to the manufacturer’s instructions, and then subjected to direct, automated sequencing (BigDye Terminator v.3.1 chemistry, Applied Biosystems, USA) in both directions using the same, internal primers as employed in PCR. The quality of each sequence was assessed based on the corresponding chromatogram, and sequences were matched to reference sequences from the GenBank database (listed in Additional file : Table S2) using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/BLAST). Chi-square test was performed using SPSS Statistics 24 software (IBM, New York, USA). […]

Pipeline specifications

Software tools BLASTN, SPSS
Application Miscellaneous
Organisms Bos taurus, Homo sapiens, Anaplasma phagocytophilum, Giardia intestinalis
Diseases Infection, Intestinal Diseases