Koehler et al. Parasites & Vectors (2017) 10:509 DOI 10.1186/s13071-017-2462-4 RESEARCH Open Access Use of a bioinformatic-assisted primer design strategy to establish a new nested PCR-based method for Cryptosporidium Anson V. Koehler1*, Pasi K. Korhonen1, Ross S. Hall1, Neil D. Young1, Tao Wang1, Shane R. Haydon2 and Robin B. Gasser1* Abstract Background: The accurate tracking of Cryptosporidium in faecal, water and/or soil samples in water catchment areas is central to developing strategies to manage the potential risk of cryptosporidiosis transmission to humans. Various PCR assays are used for this purpose. Although some assays achieve specific amplification from Cryptosporidium DNA in animal faecal samples, some do not. Indeed, we have observed non-specificity of some oligonucleotide primers in the small subunit of nuclear ribosomal RNA gene (SSU), which has presented an obstacle to the identification and classification of Cryptosporidium species and genotypes (taxa) from faecal samples. Results: Using a novel bioinformatic approach, we explored all available Cryptosporidium genome sequences for new and diagnostically-informative, multi-copy regions to specifically design oligonucleotide primers in the large subunit of nuclear ribosomal RNA gene (LSU) as a basis for an effective nested PCR-based sequencing method for the identification and/or classification of Cryptosporidium taxa. Conclusion: This newly established PCR, which has high analytical specificity and sensitivity, is now in routine use in our laboratory, together with other assays developed by various colleagues. Although the present bioinformatic workflow used here was for the specific design of primers in nuclear DNA of Cryptosporidium, this approach should be broadly applicable to many other microorganisms. Keywords: Cryptosporidium, Bioinformatics, Large subunit of nuclear ribosomal RNA gene (LSU), D8 domain, Primers, Polymerase chain reaction (PCR) Background As the oocysts and other developmental stages of dif- Animal faecal contamination in drinking water catch- ferent Cryptosporidium taxa cannot be unequivocally ment areas is a global concern, and has the potential to identified or differentiated microscopically, molecular lead to waterborne outbreaks of human gastrointestinal tools can be used for their specific and genotypic identi- diseases including cryptosporidiosis, caused by members fication and classification [6, 7]. A widely-used approach of the genus Cryptosporidium [1–5]. The identification has been nested PCR-based sequencing or restriction and classification of Cryptosporidium species and geno- fragment length polymorphism (RFLP) of particular gene types (taxa) in faecal, water and/or soil samples in water markers within the actin, cowp, hsp70 and/or small sub- catchment areas is central to assessing the risk of zoo- unit of nuclear ribosomal RNA (SSU) genes (e.g. [3, 7–9]). notic transmission of cryptosporidiosis and to develop- The sequencing of SSU has been particularly com- ing strategies to monitor and manage this risk [1–5]. monly used, because of its multi-copy nature [10], making PCR analytically sensitive, and because it con- tains diagnostically informative markers for the classi- * Correspondence: [email protected]; [email protected] fication of Cryptosporidium taxa (cf. [3, 9, 11–13]). 1 Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty Indeed, to date, more than 34 species and 40 of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010, Australia Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Koehler et al. Parasites & Vectors (2017) 10:509 Page 2 of 8 genotypes of Cryptosporidium have been classified primary PCR (~850 bp) was conducted using primers using partial SSU sequence data [3, 5, 14–17]. XF2 (forward: 5′-GGA AGG GTT GTA TTT ATT AGA Various PCR protocols are used for the amplification TAA AG-3′) and XR2 (reverse: 5′-AAG GAG TAA of regions within SSU (e.g. [8, 11, 12, 18]). When applied GGA ACA ACC TCC A-3′) (cf. [8]), followed by a sec- to faecal DNA samples from humans, most of these pro- ondary (nested) PCR (215 bp) using primers pSSUf (for- tocols appear to achieve relatively specific amplification ward: 5′-AAA GCT CGT AGT TGG ATT TCT GTT- from Cryptosporidium DNA for direct amplicon- 3′) and pSSUr (reverse: 5′-ACC TCT GAC TGT TAA sequencing. However, although not necessarily alluded ATA CRA ATG C-3′). All oligonucleotide primers (de- to in the published literature, some PCR assays do not generate nucleotide positions indicated with an Inter- achieve high specificity when applied to faecal DNA national Union of Pure and Applied Chemistry (IUPAC) samples originating from various animals, indicating that code) were synthesized by GeneWorks, Thebarton, some primer sets designed are not entirely specific for Australia. Cryptosporidium. Indeed, we have observed ‘cross-ampli- For primary PCR, the cycling protocol was: 94 °C for fication’ of SSU from DNA of some Cryptosporidium- 5 min (initial denaturation), followed by 30 cycles of 94 °C related apicomplexans (e.g. [19]) as well as selected for 45 s (denaturation), 45 °C for 2 min (annealing) and alveolates and/or dinoflagellates in animal faeces 72 °C for 1.5 min (extension), with a final extension of employing primers originally designed to Cryptospor- 72 °C for 10 min. For secondary PCR, the protocol idium spp. (Koehler et al., unpublished findings). Al- was: 94 °C for 5 min, followed by 35 cycles of 94 °C though we have designed and tested numerous new for30s,55°Cfor30sand72°Cfor30s,witha primer pairs, we did not achieve the desired level of final extension of 72 °C for 10 min. Amplicons were specificity. Other primer sets to SSU [18] were de- individually treated with ExoSAP-IT (Affymetrix, signed to improve the sensitivity of primers over the Santa Clara, CA, USA), according to the manufacturer’s leading primer set [8] by decreasing the regions tar- instructions, and then subjected to bi-directional auto- geted in the primary and secondary (nested) PCR mated sequencing (BigDye® Terminator v.3.1 chemistry, amplification steps. Here, we used a novel bioinfor- Applied Biosystems, Foster City, CA, USA) using the same matic approach to explore all available Cryptosporid- primers employed in the secondary (nested) PCR. Se- ium genome sequences (cf. [20]) for new and quence quality was verified by comparison with corre- diagnostically-informative, multi-copy regions that sponding electropherograms using the program Geneious would allow us to specifically design oligonucleotide v.9.1.4 [27]. Each SSU-derived sequence obtained was primers to establish a nested PCR-based sequencing compared by BLASTn analysis with publicly available data method with high analytical specificity and sensitivity (GenBank) (cf. [2, 3]). Based on the results, five distinct for the identification and/or classification of Crypto- groups of genomic DNA samples were formed (Additional sporidium species and genotypes. file 1: Table S1): Group 1 (well-defined ‘positive’ samples containing Methods DNA of particular Cryptosporidium taxa but without Genomic DNAs from faecal deposits, and grouping of ‘cross-amplification’): Samples (n = 20; Additional file 1: samples based on results from PCR-based sequencing of Table S1; Fig. 1) containing one of the following species: SSU C. avium, C. baileyi, C. bovis, C. canis, C. cuniculus, C. In the present study, we used a total of 332 genomic fayeri, C. felis, C. hominis, C. macropodum, C. meleagri- DNA samples from faecal samples from humans and 13 dis, C. muris, C. parvum, Cryptosporidium cf. ryanae, C. different animal species (mammals and birds) available serpentis, Cryptosporidium cf. suis, C. ubiquitum, C. from previous studies [2, 3, 21–23] or provided by col- xiaoi, Cryptosporidium sp. “deer genotype”, Cryptospor- leagues (cf. Additional file 1: Table S1). The host origin idium sp. “duck-like genotype”, Cryptosporidium cf. of each sample was known or inferred using an authori- fayeri EGK1, Cryptosporidium sp. “kangaroo genotype I”. tative field guide [24], and verified, as required, by PCR- Cryptosporidium sp. “mink genotype” or Cryptosporid- based sequencing of a region of the mitochondrial cyto- ium sp. “novel human genotype”. chrome b gene from faecal DNA (cf. [25]). Genomic Group 2 (samples with substantial cross-amplification DNA was usually extracted directly from 0.25 g of each and no evidence of the presence of Cryptosporidium): faecal sample using the PowerSoil kit (MoBio, Carlsbad, Samples (n = 13; Additional file 1: Table S1) from which CA, USA) as described previously [3]; published evi- one of the following microorganisms was amplified by dence demonstrates that this method removes PCR- PCR of SSU and sequenced: Adelina bambarooniae, Col- inhibitory components from faecal samples [26]. podella sp., Cryptosporidium struthionis (not recognized Genomic