PCR detection of Cryptosporidium parvum in environmental samples—a review of published protocols and current developments

Since 1991 more than 30 PCR protocols have been published, which show a potential to replace the current microscopic detection method for Cryptosporidium parvum in environmental samples and food. This review provides a synoptic comparison of these protocols with respect to the following features: isolation and purification of oocysts from tested matrices, elimination of free DNA, viability and infectivity assessment, release of nucleic acids, nucleic acid extraction, type of PCR (PCR, RT-PCR, internal-standard-PCR, in situ PCR, TaqMan-PCR), primary product detection, additional specificity control, secondary product detection, reported sensitivity, cross-reaction with other Cryptosporidium species, and target and sequence information such as amplicon length, primer sequences, multiple copy target, presence of strain-specific differences in the amplicon, GenBank accession numbers and gene function. The results demonstrate that problems like PCR inhibition, viability assessment, and the requirement of an extreme sensitivity have been solved. PCR assays would be most valuable to control presence-absence standards in defined matrix volumes, and the setup of such standards would very much contribute to a rapid introduction of this awaited technology into routine monitoring of environmental, water and food samples, and to a further standardization of the various protocols. It can be expected that satisfactory solutions for quantification will be found for a growing number of PCR-based assays. Systematic field evaluation and interlaboratory studies will complement our present knowledge of these methods in the near future. Received 5 May 1998/ Accepted in revised form 7 September 1998     

Detection of Cryptosporidium parvum oocysts using a microfluidic device equipped with the SUS micromesh and FITC-labeled antibody

Development of a microfluidic device equipped with micromesh for detection of Cryptosporidium parvum oocyst was reported. A micromesh consisting of 10 x 10 cavities was microfabricated on the stainless steel plate by laser ablation. Each cavity size, approximately 2.7 microm in diameter, was adopted to capture a single C. parvum oocyst. Under negative pressure operation, suspensions containing microbeads or C. parvum oocysts flowed into the microchannel. Due to strong non-specific adsorption of microbeads onto the PDMS microchannel surface during sample injection, the surface was treated with air plasma, followed by treatment with 1% sodium dodecyl sulfate (SDS) solution. This process reduced the non-specific adsorption of microbeads on the microchannel to 10% or less in comparison to a non-treated microchannel. This microfluidic device equipped with the SUS micromesh was further applied for the capture of C. parvum oocysts. Trapped C. parvum oocysts were visualized by staining with FITC-labeled anti-C. parvum oocyst antibody on a micromesh and counted under fluoroscopic observation. The result obtained by our method was consistent with that obtained by direct immunofluorescence assay coupled with immunomagnetic separation (DFA-IMS) method, indicating that the SUS micromesh is useful for counting of C. parvum oocysts. The newly designed microfluidic device exploits a geometry that allowed for the entrapment of oocysts on the micromesh while providing the rapid introduction of a series of reagents and washes through the microfluidic structure. Our data indicate that this microfluidic device is useful for high-throughput counting of C. parvum oocysts from tap water sample.     

Cryptosporidium parvum: identification of a new surface adhesion protein on sporozoite and oocyst by screening of a phage-display cDNA library

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host interaction and the molecular mechanisms involved in the pathogenesis are unknown. In this study we described a novel phage display method to identify surface adhesion proteins of C. parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum expressed on the surface of T7 phage was screened with intestinal epithelial cells (IECs) from the newborn Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were then enriched using a multi-step panning procedure. Two proteins of C. parvum were selected, one was previously reported (p23), which was an important surface adhesion protein; the other was a novel surface adherence protein (CP12). Sequence analysis showed that CP12 has a N-terminal signal peptide, a transmembrane region, a N-glycosylation site, a casein kinase II phosphorylation site and two N-myristoylation sites. Immunofluorescence assay (IFA) using antibody specific for rCP12 demonstrated that the antibody can specifically bind the surface of sporozoite and oocyst, especially apical region of sporozoite. The surface localization of CP12 and its involvement in the host-parasite interaction suggest that it may serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.     

Selection and identification of a new adhesion protein of Cryptosporidium parvum from a cDNA library by ribosome display

In this study, we described a novel display method to identify surface adhesion proteins of Cryptosporidium parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum was expressed on ribosome and selectively and specifically screened with intestinal epithelial cells (IECs) from newborn Cryptosporidium-free Holstein calves. Proteins were then enriched using a multi-step panning procedure. A new surface adherence protein of C. parvum was selected, named Cp20. Sequence analyses showed that Cp20 has a N-terminal signal peptide and four transmembrane regions. Indirect immunofluorescence assay (IFA) using an antibody specific for rCp20 demonstrated that the antibody specifically bound to the surface of sporozoites and oocysts. The recombinant plasmid pVAX1-Cp20 was constructed to examine the potential of the Cp20 gene as a target for specific preventive and therapeutic measures for cryptosporidiosis. The in vivo efficacies of the DNA vaccine was tested in BALB/c mice. The results indicated that the DNA vaccine elicited significant antibody responses and specific cellular responses when compared to control mice that received vector only or PBS. The DNA vaccine induced strong protective immune response against C. parvum and lower level of the oocysts shedding after challenge infection. This study suggested that Cp20 could serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.     

Identification and characterization of peptides that bind human ErbB-2 selected from a bacteriophage display library

The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies. The overexpression of ErbB-2 in cancer cells as well as its extracellular accessibility makes it an attractive target for the development of tumor-specific agents. In this study, random peptide bacteriophage display technology was employed to identify peptides that bound the extracellular domain of human ErbB-2. The peptide KCCYSL, most frequently occurring in the affinity-selected phage population, was chemically synthesized and characterized for its binding activities to ErbB-2. The synthetic peptide exhibited high specificity for ErbB-2 and an equilibrium dissociation constant of 30 M. Peptide binding to ErbB-2 positive human breast and prostate carcinoma cells was visualized in direct cell binding assays. In conclusion, the peptide KCCYSL has the potential to be developed into a cancer imaging or therapeutic agent targeting malignant cells overexpressing the ErbB-2 receptor.     

DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus,Scutellospora castanea

A technique has been developed to efficiently extract purified, restrictable genomic DNA from spores of different arbuscular mycorrhizal fungi in order to begin detailed investigations of the genome of the Glomales. The protocol yielded variable amounts of DNA depending on the fungal species; for Scutellospora castanea and Gigaspora rosea it reached values of 1.5–2 ng/spore. EcoRI digests of DNA from S. castanea were cloned into pUC18 and about 1000 recombinant DNA clones were obtained. Of those screened, 50 contained inserts of 500–7000 bp. Selected inserts detected DNA sequences from S. castanea spores or roots infected by this fungus, but not from nonmycorrhizal roots. This is the first report of a partial genomic library from an arbuscular mycorrhizal fungus.     

High-speed gel microelectrophoresis, a new and easy approach for detection of PCR-amplified microbial DNA from environmental and clinical samples in microgels using conventional equipment

AIMS: Microelectrophoresis allows the detection of DNA bands using minimal amounts of sample in a short time, but commonly requires the use of special equipment which is not available in all laboratories. This fact has limited the application of this technique in microbiology despite its advantages. In this work, we describe a new approach to perform gel microelectrophoresis, named high-speed gel microelectrophoresis (HSGME), and its application for rapid detection of bacteria, protozoa and viruses in clinical, vegetal and environmental samples. METHODS AND RESULTS: Aliquots of 0.4-1 microl of PCR product were loaded in 2 cm 1% agarose microgels and electrophoresed at high voltage (125 V cm(-1)) in conventional submarine horizontal mini-slabs. By using HSGME, single-DNA bands obtained after specific-PCR useful in diagnosis of different diseases caused by micro-organisms were detected in 5 min. CONCLUSIONS: HSGME is a rapid and easy procedure applicable to detection of microbial genes, which is carried out using conventional equipment and thus can be performed in any research and diagnostic laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: The performance of HSGME saves up to 90% time, material and energy costs, as well as laboratory hazardous wastes including carcinogenic agents used for visualizing DNA bands.     

Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library

A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.     

Identification of a ligand for IgG-Fc derived from a soluble peptide library based on fusion proteins secreted by S. cerevisiae

Biological libraries are important tools in the development of new peptide-based compounds. Here, we describe the use of a soluble peptide library system as a complementary tool in the field of ligand development. Random peptides were expressed in S. cerevisiae as carboxy-terminal extensions of the eukaryotic initiation factor 5a (eIF5a) and secreted into the culture supernatant. Expression and screening of this library were performed in a microwell format. As an example of this versatile approach, we describe the identification of a ligand for the human IgG-Fc fragment. Ligands binding IgG-Fc show great potential in a wide variety of applications including development of therapeutics, streamlining the large-scale purification of antibodies, and applications in diagnostic tests. We demonstrated the utility of this system. After screening only 6160 clones, we identified a ligand with the peptide sequence of TRRRTCSPPTWPRARARSTPSGCSSTGPSANRG. An affinity constant of 3.9 x 10(5) M(-1) was determined by a biosensor method. Handling and maintenance of this library is conceptually simple and highly applicable for automated high-throughput systems.     

Identification and characterisation of thirteen new microsatellites for Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis> L.) from a repeat-enriched library

A total of 13 polymorphic microsatellite markers were developed for Atlantic cod (Gadus morhua L.) from a repeat-enriched library. Polymorphism of each locus was assessed in 96 unrelated individuals from a natural population. The number of alleles per locus varied from 8 to 45. The ranges of observed and expected heterozygosity were 0.122–0.907 and 0.673–0.965, respectively. Four of the loci (Gmo-G24, Gmo-G40, Gmo-G46 and Gmo-G49) followed Hardy–Weinberg expectation. No evidence for linkage disequilibrium between pairs of loci was found in any combination of loci pairs, except between Gmo-G40 and Gmo-G43. These microsatellite markers provide useful tools for studies of population genetics, reproductive ecology and for constructing linkage maps of Atlantic cod. Jon-Ivar Westgaard and Tekle Tafese have contributed equally to the work.     

Identification of avidin and streptavidin binding motifs among peptides selected from a synthetic peptide library consisting solely of D-amino acids

Peptides consisting solely of D -amino acids (D -peptides) as opposed to their L -counterparts (L -peptides) are resistant towards proteolytic degradation in the organism and may therefore be useful in future efforts to develop new stable peptide-based drugs. Using the random synthetic peptide library technique several L - and D -peptides, capable of binding to both avidin and streptavidin, were found. The L -peptides contained the previously described HPQ/M motifis, and among the D -peptides three binding motifs could be identified, of which the most frequently found one contained an N-terminal aliphatic hydrophobic amino acid (V, L or I) and an aromatic amino acid (Y or F) on the second position. At the third position in this motif several different amino acid residues were found, although N was the most frequent. Peptides representing two of the D -motifs were synthesized as well as peptides containing the HPQ/M motifs, and their binding properties were examined. Although the D -peptides were originally selected using avidin they also inhibited binding between immobilized biotin and soluble streptavidin as well as avidin. The IC₅₀ of some of the peptides were approximately 10⁵ times higher than the IC₅₀ for biotin but some had a lower IC₅₀ than iminobiotin. The D -peptides, which were originally selected from the library using avidin, could also inhibit the binding between streptavidin and biotin. Likewise, L -peptides selected from a library screened with streptavidin, could inhibit the binding of both streptavidin and avidin to immobilized biotin. Furthermore, the D -peptide, VFSVQSGS, as well as biotin could inhibit binding of streptavidin to an immobilized L -peptide (RYHPQSGS). This indicates that the biotin-like structure mimicked by these two seemingly very different peptides may react with the same binding sites in the streptavidin molecule.     

Identification, affinity characterisation and biological interactions of lectin-like peptide-carbohydrate complexes derived from human TNF-alpha using high-resolution mass spectrometry.

A cyclic disulfide heptadecapeptide (TIP17ox; 2) derived from the lectin-like 17-amino acid domain of human tumor necrosis factor-alpha [TNF-alpha (100-116)] was synthesised and demonstrated to bind specifically to N,N-diacetylchitobiose, a disaccharide present in many glycan structures of glycoproteins. Although the TIP domain forms a loop structure in the native TNF-alpha protein, we show in this study by high-resolution ESI-FTICR mass spectrometry that a homologous linear heptadecapeptide (TIP17rd; 1) binds with comparable affinity to chitobiose, suggesting that cyclisation is not essential for carbohydrate binding. ESI-FTICR-MS was used as an efficient tool for the direct molecular characterisation of TIP peptide-carbohydrate complexes. The specific binding of the TNF-TIP domain to chitobiose and other carbohydrate motifs in glycoproteins may explain the high proteolytic stability of these peptides in biological fluids. A considerably higher proteolytic stability in human plasma was found by mass spectrometric analysis for the cyclic TIP peptide 2, compared to the linear peptide 1. Furthermore, affinity-proteomics studies using immobilised cyclic TIP peptide 2 provided the identification of specific interacting glycoproteins in plasma.     

Identification and characterization of cow's milk proteins from the rat intestinal lymph using a proteomic strategy

Food proteins were considered to be absorbed into the body after being digested to amino acids, dipeptides, and tripeptides. However, there are studies indicating that some proteins can pass through the intestinal epithelium under normal physiological conditions, perhaps not in sufficient quantities to be of nutritional importance, but in quantities that may be antigenically or biologically active. In the present study, rat intestinal lymph samples were collected using a modified lymph fistula rat model in fasting and cow's milk postprandial states. Low molecular weight proteins were enriched by ultrafiltration and differential solubilization, separated by 1D‐SDS‐PAGE, digested in‐gel based on molecular weight, and identified using nano‐LC‐MS/MS. In the postprandial rat intestinal lymph, nine bovine‐specific proteins (false discovery rate ≤1%) were identified in different molecular weight regions. Most proteins identified in lymph were highly abundant proteins in the milk, such as β‐lactoglobulin and caseins. Seven of the nine identified bovine‐specific proteins are allergens in milk. This strategy can be used to search for proteins that can enter the intestinal lymph and analyze their common features. Understanding the common features of these proteins might help to develop protein drugs taken orally, so that therapeutic proteins might embody fusion domains for cross‐barrier transport or translocation.