Specific and quantitative detection and identification of Cryptosporidium hominis andC. parvum in clinical and environmental samples

Abstract

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Molecular characterisation of a disseminated Cryptosporidium infection in a Koi carp (Cyprinus carpio)

“…Isolates positive at the 18S locus were also analysed at the actin locus using a hemi-nested PCR optimized for amplification of piscine-derived Cryptosporidium species, as previously described (Koinari et al, 2013). The isolate was also also screened using a C. parvum and C. hominis specific qPCR at a unique Cryptosporidium specific gene (Clec) coding for a novel mucin-like glycoprotein that contains a C-type lectin domain (CTLD) previously described (Yang et al, 2013). This was done to determine if there was a mixed infection with C. parvum and/or C. hominis in the fish.…”

“…Physical separation of sample preparation and amplification areas was practiced to prevent contamination of test samples by PCR products. The amplified DNA fragments from the secondary PCR products were separated by gel electrophoresis and purified for sequencing using an in-house filter tip-based method without any further purification as previously described (Yang et al, 2013).…”

First report of Trypanosoma vegrandis in koalas (Phascolarctos cinereus)

“…The PCR product from these external primers was run on a 2% agarose gel stained with SYBR safe (Invitrogen, USA). The gel band was excised and purified using an in-house filter tip method as previously described [13] and 1 µl of gel-purified PCR was used in a secondary reaction with internal primers TVIF and TVIR [5]. In order to detect co-infections with other trypanosome species, all samples that were positive for T. vegrandis using the T. vegrandis-specific primers were also screened by nested PCR using generic Trypanosoma sp.…”

“…All positive T. vegrandis-specific PCR products were purified using the filter tip method previously described [13] and sequenced in both directions using the T. vegrandisspecific internal forward TVIF and reverse TVIR primers [5] with an ABI Prism TM Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, California, U.S.A.) on an Applied Biosystem 3730 DNA Analyzer. For amplicons generated using the universal trypanosome primers, sequencing was conducted using the internal PCR primers SSU561F…”

Greater intensity and frequency of Cryptosporidium and Giardia oocyst shedding beyond the neonatal period is associated with reductions in growth, carcase weight and dressing efficiency in sheep

“…An internal amplification control (IAC) which consisted of a fragment of a coding region from Jembrana Disease Virus cloned into a pGEM-T vector (Promega, USA), was used as previously described (Yang et al, 2013). All Cryptosporidium positives were screened using a C. parvum and C. hominis specific qPCR at a unique Cryptosporidium specific gene (Clec) that codes for a novel mucin-like glycoprotein that contains a C-type lectin domain (CTLD) previously described (Morgan et al, 1996;Yang et al, 2009;Bhalchandra et al, 2013).…”