Use of DNA melting simulation software for in silico diagnostic assay design: targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes

BACKGROUND: DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium amplicons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria. RESULTS: The SYTO9 melting curve profiles of three species of Naegleria and two species of Cryptosporidium were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated in vitro. Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts in vitro. CONCLUSION: Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for different species of Naegleria and Cryptosporidium and the complex melting profiles generated in vitro using SYTO9 verified that the complex melting profile of PCR amplicons was solely the result of DNA dissociation. Other data outputs from these simulations were also used to identify the melting domains that contributed to the observed melting peaks for each of the different PCR amplicons

It's official - Cryptosporidium is a gregarine: What are the implications for the water industry?

Parasites of the genus Cryptosporidium are a major cause of diarrhoea and ill-health in humans and animals and are frequent causes of waterborne outbreaks. Until recently, it was thought that Cryptosporidium was an obligate intracellular parasite that only replicated within a suitable host, and that faecally shed oocysts could survive in the environment but could not multiply. In light of extensive biological and molecular data, including the ability of Cryptosporidium to complete its life cycle in the absence of a host and the production of novel extracellular stages, Cryptosporidium has been formally transferred from the Coccidia, to a new subclass, Cryptogregaria, with gregarine parasites. In this review, we discuss the close relationship between Cryptosporidium and gregarines and discuss the implications for the water industry

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R2≥0.999) and faecal samples (R2≥0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that the overall cost (consumables and labour) of droplet digital PCR was two times higher than quantitative PCR. Using droplet digital PCR to precisely quantify standard dilutions used for high-throughput and cost-effective amplifications by quantitative PCR would be one way to combine the advantages of the two technologies

Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community

Giardia duodenalis isolates recovered from humans and dogs living in the same locality in a remote tea-growing community of northeast India were characterized at 3 different loci; the SSU-rDNA, elongation factor 1-alpha (ef1-α) and triose phosphate isomerase (tpi) gene. Phylogenetic analysis of the SSU-rDNA and ef1-α genes provided poor genetic resolution of the isolates within various assemblages, stressing the importance of using multiple loci when inferring genotypes to Giardia. Analysis of the tpi gene provided better genetic resolution and placed canine Giardia isolates within the genetic groupings of human isolates (Assemblages A and B). Further evidence for zoonotic transmission was supported by epidemiological data showing a highly significant association between the prevalence of Giardia in humans and presence of a Giardia-positive dog in the same household (odds ratio 3.01, 95% CI, 1.11, 8.39, P = 0.0000)

Organoids and bioengineered intestinal models: Potential solutions to the Cryptosporidium culturing dilemma

Cryptosporidium is a major cause of severe diarrhea-related disease in children in developing countries, but currently no vaccine or effective treatment exists for those who are most at risk of serious illness. This is partly due to the lack of in vitro culturing methods that are able to support the entire Cryptosporidium life cycle, which has led to research in Cryptosporidium biology lagging behind other protozoan parasites. In vivo models such as gnotobiotic piglets are complex, and standard in vitro culturing methods in transformed cell lines, such as HCT-8 cells, have not been able to fully support fertilization occurring in vitro. Additionally, the Cryptosporidium life cycle has also been reported to occur in the absence of host cells. Recently developed bioengineered intestinal models, however, have shown more promising results and are able to reproduce a whole cycle of infectivity in one model system. This review evaluates the recent advances in Cryptosporidium culturing techniques and proposes future directions for research that may build upon these successes

Identification of polymorphic genes for use in assemblage B genotyping assays through comparative genomics of multiple assemblage B Giardia duodenalis isolates

Giardia duodenalis assemblage B is potentially a zoonotic parasite. The characterisation and investigation of isolates has been hampered by greater genetic diversity of assemblage B, limiting the application and utility of current genotyping loci. Since whole genome sequencing is the optimal high-throughput method for gene identification, the present study sequenced assemblage B isolate BAH15c1 and compared the sequence to the draft GS references to identify polymorphic genes for potential use in genotyping assays. The majority of the genome sequence was conserved between the two isolates, producing 508 contigs of 10.4 Mb with 4968 genes. Seventy polymorphic genes for potential use in genotyping assays were identified ranging in variation from elongation factor 1 α, which was the most conserved, through to triose phosphate isomerase, which was the most variable

Molecular characterisation of Cryptosporidium and Giardia in cats (Felis catus) in Western Australia

Little is known of the prevalence of Cryptosporidium and Giardia in domestic cats in Western Australia and their potential role as zoonotic reservoirs for human infection. In the present study, a total of 345 faecal samples from four different sources were screened for the presence of Cryptosporidium and Giardia by PCR and genotyped by sequence analysis. Oocyst numbers and cyst numbers for Cryptosporidium and Giardia respectively were also determined using quantitative PCR assays. Cryptosporidium and Giardia were detected in 9.9% (95% CI 6.7-13.0) and 10.1% (95% CI 7.0-13.3) of cats in Western Australia respectively. Sequence analysis at the 18S rRNA locus identified five Cryptosporidium species/genotypes; C. felis (n = 8), C. muris (n = 1), C. ryanae (n = 1), Cryptosporidium rat genotype III (n = 5) and a novel genotype most closely related to Cryptosporidium rat genotype III in one isolate. This is the first report of C. ryanae and Cryptosporidium rat genotype III in cats. For Giardia, assemblage F the most commonly identified species, while only 1 assemblage sequence was detected. Since most human cases of cryptosporidiosis are caused by C. parvum and C. hominis and human cases of giardiasis are caused by G. duodenalis assemblage A and B, the domestic cats in the present study are likely to be of low zoonotic risk to pet owners in Perth. Risk analyses identified that elderly cats (more than 6 years) were more prone to Cryptosporidium and Giardia infections than kittens (less than 6 months) (P = 0.009). Clinical symptoms were not associated with the prevalence of Cryptosporidium and Giardia infections in cats

Molecular and phylogenetic analysis of Cryptosporidium muris from various hosts

Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris; (1) C. muris genotype A; comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation

Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus

Samples of DNA from a panel of Giardia isolated from humans and animals in Europe and shown previously to consist of 2 major genotypes–‘Polish’ and ‘Belgian’–have been compared with human-derived Australian isolates chosen to represent distinct genotypes (genetic groups I–IV) defined previously by allozymic analysis. Homologous 0·52 kilobase (kb) segments of 2 trophozoite surface protein genes (tsa417 and tsp11, both present in isolates belonging to genetic groups I and II) and a 1·2 kb segment of the glutamate dehydrogenase (gdh) gene were amplified by the polymerase chain reaction (PCR) and examined for restriction fragment length polymorphisms (RFLPs). Of 21 ‘Polish’ isolates that were tested, all yielded tsa417-like and tsp11-like PCR products that are characteristic of genetic groups I or II (15 and 6 isolates respectively) in a distinct assemblage of G. intestinalis from Australia (Assemblage A). Conversely, most of the 19 ‘Belgian’ isolates resembled a second assemblage of genotypes defined in Australia (Assemblage B) which contains genetic groups III and IV. RFLP analysis of gdh amplification products showed also that ‘Polish’ isolates-were equivalent to Australian Assemblage A isolates (this analysis does not distinguish between genetic groups I and II) and that ‘Belgian’ isolates were equivalent to Australian Assemblage B isolates. Comparison of nucleotide sequences determined for a 690 base-pair portion of the gdh PCR products revealed ≥ 99·0% identity between group I and group II (Assemblage A/‘Polish’) genotypes, 88·3–89·7% identity between Assemblage A and Assemblage B genotypes, and ≥ 98·4% identity between various Assemblage B/‘Belgian’ genotypes. The results confirm that the G. duodenalis isolates examined in this study (inclusive of G. intestinalis from humans) can be divided into 2 major genetic clusters: Assemblage A (= ‘Polish’ genotype) containing allozymically defined groups I and II, and Assemblage B (= ‘Belgian’ genotype) containing allozymically defined groups III and IV and other related genotypes

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.

We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 microL of crude lysate in under 30 min for the entire process