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Multiplex PCR for the detection and quantification of zoonotic taxa of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels
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Multiplex PCR for the detection and quantification of key zoonotic genotypes of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels Q6
Marianna Marangi a, b, *, Annunziata Giangaspero a, Vita Lacasella a, Antonio Lonigro c, Robin B. Gasser b, * a b c
Department of Science of Agriculture, Food and Environment, University of Foggia, 71121 Foggia, Italy Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Victoria 3010, Australia Department of Agricultural and Environmental Science, University of Bari, 70126 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2014 Accepted 5 January 2015 Available online xxx
Giardia duodenalis, Cryptosporidium parvum and Toxoplasma gondii are important parasitic protists linked to water- and food-borne diseases. The accurate detection of these pathogens is central to the diagnosis, tracking, monitoring and surveillance of these protists in humans, animals and the environment. In this study, we established a multiplex real-time PCR (qPCR), coupled to high resolution melting (HRM) analysis, for the specific detection and quantification of each G. duodenalis (assemblage A), C. parvum and T. gondii (Type I). Once optimised, this assay was applied to the testing of samples (n ¼ 232) of treated wastewater and mussels (Mytilus galloprovincialis). Of 119 water samples, 28.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens; of 113 mussel samples 66.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens, and 13.2% were test-positive for only T. gondii. The specificity of all amplicons produced was verified by direct sequencing. The oo/cysts numbers (per 5 ml of DNA sample) ranged from 10 to 64. The present multiplex assay achieved an efficiency of 100% and a R2 value of >0.99. Current evidence indicates that this assay provides a promising tool for the simultaneous detection and quantitation of three key protists. © 2015 Published by Elsevier Ltd.
Keywords: Multiplex PCR Protists Giardia Cryptosporidium Toxoplasma
1. Introduction Giardia, Cryptosporidium and Toxoplasma are protistan pathogens that have received considerable attention because of their roles in water- and/or food-borne diseases [1,2]. Giardia and Cryptosporidium spp. are well-known causative agents of enteric diseases worldwide, particularly in young, elderly and immunocompromised or -suppressed subjects [3]. Giardia duodenalis (assemblage A) and Cryptosporidium parvum are considered to be of prime zoonotic importance [4,5]. Toxoplasma gondii is a protist that usually causes asymptomatic infection in immune-competent people, but can induce extraintestinal disease in immuno-
Q1
* Corresponding authors. Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Victoria 3010, Australia. Tel.: þ39 0881 589227; fax: þ39 088 589501. E-mail addresses: [email protected] (M. Marangi), robinbg@unimelb. edu.au (R.B. Gasser).
compromised individuals, often leading to abortion in pregnant women and involvement of the central nervous system [6]. T. gondii (Type I) is considered most pathogenic [7,8]. Due to the resilience of infective stages (cysts and oocysts) in the environment, particularly in water, these protists have been frequently reported as the cause of food- or waterborne outbreaks, worldwide [9e11]. Since the public concern about water and food safety has increased significantly in the last decade, molecular tools are used to confirm cases of giardiasis, cryptosporidiosis and toxoplasmosis, to track transmission in outbreak situations and also to monitor the success of control. Advanced molecular tools, such as PCR-based methods, have been developed as epidemiological tools to improve the diagnosis, the rapid detection, identification and differentiation of protists of water and/or food safety concern [12]. Some real-time or quantitative (q)PCR techniques have been set up for Giardia, Cryptosporidium or Toxoplasma [5,13e17], and beta-giardin, COWP and B1 genes are the genetic markers commonly employed to detect and/
http://dx.doi.org/10.1016/j.mcp.2015.01.001 0890-8508/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Marangi M, et al., Multiplex PCR for the detection and quantification of key zoonotic genotypes of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.01.001
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or genetically identify these protists [18e21]. Due to the lack of a multiplex qPCR for the simultaneous detection and quantification of water- and food-borne protozoans, we established a practical and cost effective multiplex qPCR, coupled to high resolution melting (HRM) analysis, for the specific quantitative detection of G. duodenalis (assemblage A), C. parvum and T. gondii (Type I). 2. Materials and methods 2.1. Genomic DNA samples Genomic DNAs were isolated using Nucleospin tissue kit (MachereyeNagel, Germany) from 119 wastewater samples collected from four water treatment plants using different processing methods (i.e. sand, membrane-bioreactor, plug-flow reactor and membrane ultrafiltration, respectively) and collected and processed as described previously [22], and also from 113 mussel (Mytilus galloprovincialis) samples (53 from Turkey and 60 from Italy) available from previous studies [23,24]. 2.2. Design and assessment of oligonucleotide primers for use in multiplex qPCR Three primer pairs (forward and reverse) were designed to the genes b-giardin (G. duodenalis assemblage A; accession no. X85958; [25]), COWP (C. parvum; accession no. Z22537; [26]) and the repetitive gene B1 (T. gondii (Type I); accession no. AF179871; [27]) (see Table 1). The specificity of each primer was first predicted in silico by comparing their sequences against all publicly available nucleotide sequences using BLASTn software [28]; no primer sequence matched any sequence of any taxon other than that to which it was designed. Then, each of the three primer pairs was tested in qPCR (same conditions as final protocol; see below) against genomic DNAs representing each of the three target taxa, and amplicons (following treatment with ExoI-FastAP, Fermentas) directly sequenced (BygDye Terminator v. 3.1, Applied Biosystems) using homologous primers employed in each respective PCR. Primer pairs were then combined into one multiplex reaction (described below), and tested against the same genomic DNAs (representing each of the three taxa being tested for); amplicons of the expected sizes (on agarose gels) were produced only from genomic DNAs of homologous taxa, and no additional spurious amplicons were detectable on agarose gels; direct sequencing verified the specificity of all amplicons produced. 2.3. Multiplex qPCR assay The three primer pairs GGL-GGR, CRYINT2D-CRY9D and ToxB41f-ToxB169r (Table 1) were used in one reaction for the specific and simultaneous amplification of G duodenalis (assemblage A), C. parvum and T. gondii (Type I) from DNA samples. PCR was carried out in a volume of 20 ml using a standard buffer and the fluorescent dye EvaGreen® (BioRad, USA), 0.5 mM (final
concentration) of each forward and reverse primer. Samples without genomic DNA (no-DNA controls) were included in each PCR run. Also included in each run was a serial titration (1010, 109, 108, 107, 106, 105, 104, 103, 102 and 10 copies) of b-giardin, COWP and the B1 gene (cloned into the vector pEX-A; Eurofins) of each G. duodenalis (assemblage A), C. parvum and T. gondii (Type I), respectively, as positive reference controls (to provide standard curves). Cycling conditions in a CFX-96 thermocycler (BioRad) were: initial denaturation at 98 C for 2 min, followed by amplification for 35 cycles of 98 C for 5 s and 58 C for 15 s. Fluorescence data were collected at the end of each cycle as a single acquisition. To verify their specificity, amplicons were subjected to melting-curve analysis (75e95 C at 0.5 C/5 s) in CFX-96 thermocycler (BioRad, USA) using Precision Melt Analysis software v.1.2. The melting temperature (Tm) was interpolated from the normalised data as the temperature at 50% fluorescence. Tm and standard deviation (SD) was recorded for each reference (positive) control. Test-positive samples were identified on basis of a single melt-peak, which was consistent with that of the homologous control for each PCR. The melting peaks were 85 C for G. duodenalis (assemblage A), 75 C for C. parvum and 80 C for T. gondii (Type I). Again, all amplicons produced were directly sequenced to verify their specificity. Any suspected inhibition in PCR, likely linked to faecal constituents (e.g., humic acids, phenolic compounds and/or polysaccharides), was explored in spiking experiments. To do this, aliquots (1 ml) of selected samples that were test-negative by PCR were spiked with a limiting amount (10 copies) of each reference control DNA representing each of the three target taxa. For each taxon, the number of gene copies (per ml) was calculated by relating the Ct mean value of each sample to the standard curve for the corresponding reference control. The number of cysts or oocysts in each sample (5 ml DNA aliquot) was calculated, assuming that the bgiardin, COWP and B1 genes have one, five and 35 copies in the genomes of G. duodenalis, C. parvum and T. gondii, respectively [27,29e31]. 2.4. Assessing assay performance First, PCR efficiency (E) was calculated according to the equation: E ¼ 10 1/slope 1 [32]. An E value of between 90% and 110% and a correlation (R2) of <1 value were considered acceptable. Second, the “analytical” sensitivity of the multiplex qPCR was established using 10-fold serial dilutions (from 1010 to 10 copies/ml) of the (cloned) reference (positive) controls, which were each subjected (in triplicate) to PCR amplification and subsequent HRM analysis; the mean value of the threshold cycle (Ct) was plotted against the logarithm of gene copies per ml. Three standard curves were produced by a linear regression; the range of linearity and the lowest detectable number of gene copies detectable were estimated from each standard curve. Third, variation of test results within and among assays were assessed by testing three replicates of control samples (for 107, 105 and 10 copies) three times on different days and expressed as coefficients of variation (CV),
Table 1 Genes to which primers were designed and their locations in reference sequences (accession number) and predicted amplicon size upon PCR. Protist
Gene
Primer designation and sequence
Location (nucleotide positions)
Amplicon size (bp)
Accession number
Reference
Giardia duodenalis
b-giardin
X85958
[25]
COWP
315
Z22537
[26]
Toxoplasma gondii
B1
1421e1440 1571e1591 1110e1134 1446e1470 41e63 148e169
171
Cryptosporidium parvum
GGL: 50 -AAGTGCGTCAACGAGCAGCT-30 (forward) GGR: 50 -TTAGTGCTTTGTGACCATCGA-30 (reverse) CRYINT2D: 50 -TTTGTTGAAGARGGAAATAGATGTG-30 (forward) CRY2D: 50 -GGACKGAAATRCAGGCATTATCYTG-30 (forward) TOXB41F: 50 -TCGAAGCTGAGATGCTCAAAGTC-30 (forward) TOXB169R: 50 -AATCCACGTCTGGGAAGAACTC-30
129
AF179871
[27]
Please cite this article in press as: Marangi M, et al., Multiplex PCR for the detection and quantification of key zoonotic genotypes of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.01.001
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calculated using the formula: CV ¼ s(Ct)/m(Ct), as described in the manufacturer's protocol (BioRad). 3. Results and discussion First, we assessed the performance of the qPCR assay. An amplification efficiency (E) of 100.7% was achieved, with a slope of 3305 (R2 ¼ 0.999). The linearity of amplification from 1010 to 10 copies per ml was regarded as acceptable, and a minimum of 10 gene copies was detected for each of the three target protists. Intraand inter-assay variabilities were estimated at 1.6e1.7% and 0.90e0.95%, respectively. We then applied the assay to test 232 wastewater and mussel DNA samples (Table 2). In total, 34 of the 119 (28.6%) water samples were test-positive for either C. parvum or G. duodenalis or both protists; 10 (8.4%) of all 119 samples were test-positive for G. duodenalis, 18 (15.1%) for C. parvum, and 6 (5%) for G. duodenalis and C. parvum. All 119 samples were test-negative for T. gondii. In test-positive samples, the numbers of oocysts/cysts were estimated at 10 to 56 for C. parvum and 7 to 118 for G. duodenalis. Subsequently, we tested the mussel DNA samples. Seven of the 53 (13.2%) mussel DNA samples from Turkey were test-positive for T. gondii. All of these samples were test-negative for G. duodenalis and C. parvum. In addition, 40 of the 60 (66.6%) samples from Italy were test-positive for one or more target protists. Specifically, 26 (43.3%) of the 60 samples were test-positive for C. parvum, six (10%) for G. duodenalis and 8 (13.3%) for both protists; all 113 samples were test-negative for T. gondii. For test-positive mussel samples, the numbers of oocysts/cysts were estimated at 10 to 64 for C. parvum, 4 to 78 for G. duodenalis (Italy) and 6 to 30 for T. gondii (Turkey) per 5 ml of genomic DNA tested. Finally, the sequencing of wastewaterand mussel-DNA derived amplicons confirmed the specific identity of G. duodenalis assemblage A (n ¼ 24), C. parvum (n ¼ 36) and T. gondii Type I (n ¼ 7). This multiplex qPCR for the simultaneous detection of G. duodenalis (assemblage A), C. parvum and T. gondii (Type I) shows promise for use in epidemiological surveys and could assist in the monitoring and tracking disease linked to food and waterborne outbreaks. In the last decade, numerous molecular diagnostic assays for viruses and bacteria have been developed for use in diagnostic laboratories, and are now being increasingly established for a range of enteric protozoa [12,33]. Although many qPCR techniques have been developed [34,35], some of them have limitations, in terms of the cost and performance of particular dyes in
Table 2 The qPCR results for water and mussel samples tested in this study. Protists
qPCR results Number of test-positive samples/total number of samples tested Water (Italy)
Mussels (Italy) Mussels Total (Turkey) numbers (%)
Giardia duodenalis 10/119 (8.4%) 6/60 (10%) Cryptosporidium 18/119 (15.1%) 26/60 (43.3%) parvum Toxoplasma gondii 0 0 G. duodenalis & C. parvum G. duodenalis & T. gondii C. parvum & T. gondii Totals (%)
0 0
16/179 (8.9%) 44/179 (24.5%) 7/53 (13.2%) 14/179 (7.8%)
6/119 (5%)
8/60 (13.3%)
7/53 (13.2%) 0
0
0
0
0
0
0
0
0
7/53 (13.2%)
81/232 (34.9%)
34/119 (28.6%) 40/60 (66.6%)
3
PCR. For instance TaqMan probes [36] are expensive [37] or dyes, such as SyberGreen® may inhibit PCR [38], which reduces reproducibility and/or analytical sensitivity. In contrast, EvaGreen® seems to be more suitable for routine multiplex qPCR applications, as it has less inhibitory effect and lacks dye-redistribution [39,40]. The assay established here is inexpensive (costs approximately $1.50 per sample), takes 1.3 h to perform, and is quantitative and sensitive. Current evidence obtained by direct sequencing of a total of 67 amplicons indicates that the present assay should achieve specific amplification from G. duodenalis assemblage A (n ¼ 24), C. parvum (n ¼ 36) and T. gondii Type I (n ¼ 7) from wastewater and mussel samples with potential for transmission to humans, provided that cysts/oocysts of these pathogens in such samples are viable and infective. However, given the nature and extent of the different genomic DNAs that might occur in these two types of biological matrices (i.e. wastewater and mussel e a filter-feeder), such as those potentially derived from an array of viruses, bacteria, fungi, protists and/or other microorganisms or organisms (whose genomes might not yet have been sequenced), it will be crucial to keep monitoring the specificity of the qPCR (and its primers and conditions) by direct sequencing or mutation scanning [41] of amplicons produced using the three primer sets employed herein. Although melting profiles provide an indication of specificity, the qualitative analysis of amplicons by electrophoretic mutation scanning or sequencing will continue to be critical during the deployment of the present qPCR assay. This assay also could be a useful tool for the simultaneous detection and quantitation of the three zoonotic protists in various other biological matrices (such as soil, vegetables and/or fruits), but its extension to such matrices (also representing samples from different geographical origins) would require continual verification of assay specificity and sensitivity. Acknowledgements This work was partly supported by a grant from PON Puglia ERDF 2007e2013, Axis I, Line 1.2. Accordo di Programma Quadro. Reti di Laboratori Pubblici di Ricerca. Progetto L.A.I.F.F. (codice n. 47), Italy, and by a grant from PON REC 01_01480 IN.TE.R.R.A. (Technology and process innovations for irrigation reuse of treated municipal and agro-industrial wastewaters to achieve sustainable water resources management), Bari, Italy. The authors thank Dr Tiziana Caradonna for technical assistance. RBG's research was supported by the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC) of Australia, Melbourne Water Corporation, and Yourgene Bioscience; it was also supported by a Victorian Life Sciences Computation Initiative (VLSCI; grant number VR0007) on its Peak Computing Facility at the University of Melbourne, an initiative of the Victorian Government. References [1] Karanis P, Kourenti C, Smith H. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J Water Health 2007;5:1e38. [2] Dorny P, Praet N, Deckers N, Gabriel S. Emerging food-borne parasites. Vet Parasitol 2009;163:196e206. [3] Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev 2013;26:115e34. [4] Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011;24:110e40. S. Zoonotic potential of Giardia. Int J Parasitol 2013;43:943e56. [5] Ryan U, Caccio [6] Barratt JL, Harkness J, Marriott D, Ellis JT, Stark D. Importance of nonenteric protozoan infections in immunocompromised people. Clin Microbiol Rev 2010;23:795e836.
Please cite this article in press as: Marangi M, et al., Multiplex PCR for the detection and quantification of key zoonotic genotypes of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.01.001
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Multiplex PCR for the detection and quantification of key zoonotic genotypes of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels Q6
Marianna Marangi a, b, *, Annunziata Giangaspero a, Vita Lacasella a, Antonio Lonigro c, Robin B. Gasser b, * a b c
Department of Science of Agriculture, Food and Environment, University of Foggia, 71121 Foggia, Italy Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Victoria 3010, Australia Department of Agricultural and Environmental Science, University of Bari, 70126 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2014 Accepted 5 January 2015 Available online xxx
Giardia duodenalis, Cryptosporidium parvum and Toxoplasma gondii are important parasitic protists linked to water- and food-borne diseases. The accurate detection of these pathogens is central to the diagnosis, tracking, monitoring and surveillance of these protists in humans, animals and the environment. In this study, we established a multiplex real-time PCR (qPCR), coupled to high resolution melting (HRM) analysis, for the specific detection and quantification of each G. duodenalis (assemblage A), C. parvum and T. gondii (Type I). Once optimised, this assay was applied to the testing of samples (n ¼ 232) of treated wastewater and mussels (Mytilus galloprovincialis). Of 119 water samples, 28.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens; of 113 mussel samples 66.6% were test-positive for G. duodenalis, C. parvum and/or both pathogens, and 13.2% were test-positive for only T. gondii. The specificity of all amplicons produced was verified by direct sequencing. The oo/cysts numbers (per 5 ml of DNA sample) ranged from 10 to 64. The present multiplex assay achieved an efficiency of 100% and a R2 value of >0.99. Current evidence indicates that this assay provides a promising tool for the simultaneous detection and quantitation of three key protists. © 2015 Published by Elsevier Ltd.
Keywords: Multiplex PCR Protists Giardia Cryptosporidium Toxoplasma
1. Introduction Giardia, Cryptosporidium and Toxoplasma are protistan pathogens that have received considerable attention because of their roles in water- and/or food-borne diseases [1,2]. Giardia and Cryptosporidium spp. are well-known causative agents of enteric diseases worldwide, particularly in young, elderly and immunocompromised or -suppressed subjects [3]. Giardia duodenalis (assemblage A) and Cryptosporidium parvum are considered to be of prime zoonotic importance [4,5]. Toxoplasma gondii is a protist that usually causes asymptomatic infection in immune-competent people, but can induce extraintestinal disease in immuno-
Q1
* Corresponding authors. Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Victoria 3010, Australia. Tel.: þ39 0881 589227; fax: þ39 088 589501. E-mail addresses: [email protected] (M. Marangi), robinbg@unimelb. edu.au (R.B. Gasser).
compromised individuals, often leading to abortion in pregnant women and involvement of the central nervous system [6]. T. gondii (Type I) is considered most pathogenic [7,8]. Due to the resilience of infective stages (cysts and oocysts) in the environment, particularly in water, these protists have been frequently reported as the cause of food- or waterborne outbreaks, worldwide [9e11]. Since the public concern about water and food safety has increased significantly in the last decade, molecular tools are used to confirm cases of giardiasis, cryptosporidiosis and toxoplasmosis, to track transmission in outbreak situations and also to monitor the success of control. Advanced molecular tools, such as PCR-based methods, have been developed as epidemiological tools to improve the diagnosis, the rapid detection, identification and differentiation of protists of water and/or food safety concern [12]. Some real-time or quantitative (q)PCR techniques have been set up for Giardia, Cryptosporidium or Toxoplasma [5,13e17], and beta-giardin, COWP and B1 genes are the genetic markers commonly employed to detect and/
http://dx.doi.org/10.1016/j.mcp.2015.01.001 0890-8508/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Marangi M, et al., Multiplex PCR for the detection and quantification of key zoonotic genotypes of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.01.001
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or genetically identify these protists [18e21]. Due to the lack of a multiplex qPCR for the simultaneous detection and quantification of water- and food-borne protozoans, we established a practical and cost effective multiplex qPCR, coupled to high resolution melting (HRM) analysis, for the specific quantitative detection of G. duodenalis (assemblage A), C. parvum and T. gondii (Type I). 2. Materials and methods 2.1. Genomic DNA samples Genomic DNAs were isolated using Nucleospin tissue kit (MachereyeNagel, Germany) from 119 wastewater samples collected from four water treatment plants using different processing methods (i.e. sand, membrane-bioreactor, plug-flow reactor and membrane ultrafiltration, respectively) and collected and processed as described previously [22], and also from 113 mussel (Mytilus galloprovincialis) samples (53 from Turkey and 60 from Italy) available from previous studies [23,24]. 2.2. Design and assessment of oligonucleotide primers for use in multiplex qPCR Three primer pairs (forward and reverse) were designed to the genes b-giardin (G. duodenalis assemblage A; accession no. X85958; [25]), COWP (C. parvum; accession no. Z22537; [26]) and the repetitive gene B1 (T. gondii (Type I); accession no. AF179871; [27]) (see Table 1). The specificity of each primer was first predicted in silico by comparing their sequences against all publicly available nucleotide sequences using BLASTn software [28]; no primer sequence matched any sequence of any taxon other than that to which it was designed. Then, each of the three primer pairs was tested in qPCR (same conditions as final protocol; see below) against genomic DNAs representing each of the three target taxa, and amplicons (following treatment with ExoI-FastAP, Fermentas) directly sequenced (BygDye Terminator v. 3.1, Applied Biosystems) using homologous primers employed in each respective PCR. Primer pairs were then combined into one multiplex reaction (described below), and tested against the same genomic DNAs (representing each of the three taxa being tested for); amplicons of the expected sizes (on agarose gels) were produced only from genomic DNAs of homologous taxa, and no additional spurious amplicons were detectable on agarose gels; direct sequencing verified the specificity of all amplicons produced. 2.3. Multiplex qPCR assay The three primer pairs GGL-GGR, CRYINT2D-CRY9D and ToxB41f-ToxB169r (Table 1) were used in one reaction for the specific and simultaneous amplification of G duodenalis (assemblage A), C. parvum and T. gondii (Type I) from DNA samples. PCR was carried out in a volume of 20 ml using a standard buffer and the fluorescent dye EvaGreen® (BioRad, USA), 0.5 mM (final
concentration) of each forward and reverse primer. Samples without genomic DNA (no-DNA controls) were included in each PCR run. Also included in each run was a serial titration (1010, 109, 108, 107, 106, 105, 104, 103, 102 and 10 copies) of b-giardin, COWP and the B1 gene (cloned into the vector pEX-A; Eurofins) of each G. duodenalis (assemblage A), C. parvum and T. gondii (Type I), respectively, as positive reference controls (to provide standard curves). Cycling conditions in a CFX-96 thermocycler (BioRad) were: initial denaturation at 98 C for 2 min, followed by amplification for 35 cycles of 98 C for 5 s and 58 C for 15 s. Fluorescence data were collected at the end of each cycle as a single acquisition. To verify their specificity, amplicons were subjected to melting-curve analysis (75e95 C at 0.5 C/5 s) in CFX-96 thermocycler (BioRad, USA) using Precision Melt Analysis software v.1.2. The melting temperature (Tm) was interpolated from the normalised data as the temperature at 50% fluorescence. Tm and standard deviation (SD) was recorded for each reference (positive) control. Test-positive samples were identified on basis of a single melt-peak, which was consistent with that of the homologous control for each PCR. The melting peaks were 85 C for G. duodenalis (assemblage A), 75 C for C. parvum and 80 C for T. gondii (Type I). Again, all amplicons produced were directly sequenced to verify their specificity. Any suspected inhibition in PCR, likely linked to faecal constituents (e.g., humic acids, phenolic compounds and/or polysaccharides), was explored in spiking experiments. To do this, aliquots (1 ml) of selected samples that were test-negative by PCR were spiked with a limiting amount (10 copies) of each reference control DNA representing each of the three target taxa. For each taxon, the number of gene copies (per ml) was calculated by relating the Ct mean value of each sample to the standard curve for the corresponding reference control. The number of cysts or oocysts in each sample (5 ml DNA aliquot) was calculated, assuming that the bgiardin, COWP and B1 genes have one, five and 35 copies in the genomes of G. duodenalis, C. parvum and T. gondii, respectively [27,29e31]. 2.4. Assessing assay performance First, PCR efficiency (E) was calculated according to the equation: E ¼ 10 1/slope 1 [32]. An E value of between 90% and 110% and a correlation (R2) of <1 value were considered acceptable. Second, the “analytical” sensitivity of the multiplex qPCR was established using 10-fold serial dilutions (from 1010 to 10 copies/ml) of the (cloned) reference (positive) controls, which were each subjected (in triplicate) to PCR amplification and subsequent HRM analysis; the mean value of the threshold cycle (Ct) was plotted against the logarithm of gene copies per ml. Three standard curves were produced by a linear regression; the range of linearity and the lowest detectable number of gene copies detectable were estimated from each standard curve. Third, variation of test results within and among assays were assessed by testing three replicates of control samples (for 107, 105 and 10 copies) three times on different days and expressed as coefficients of variation (CV),
Table 1 Genes to which primers were designed and their locations in reference sequences (accession number) and predicted amplicon size upon PCR. Protist
Gene
Primer designation and sequence
Location (nucleotide positions)
Amplicon size (bp)
Accession number
Reference
Giardia duodenalis
b-giardin
X85958
[25]
COWP
315
Z22537
[26]
Toxoplasma gondii
B1
1421e1440 1571e1591 1110e1134 1446e1470 41e63 148e169
171
Cryptosporidium parvum
GGL: 50 -AAGTGCGTCAACGAGCAGCT-30 (forward) GGR: 50 -TTAGTGCTTTGTGACCATCGA-30 (reverse) CRYINT2D: 50 -TTTGTTGAAGARGGAAATAGATGTG-30 (forward) CRY2D: 50 -GGACKGAAATRCAGGCATTATCYTG-30 (forward) TOXB41F: 50 -TCGAAGCTGAGATGCTCAAAGTC-30 (forward) TOXB169R: 50 -AATCCACGTCTGGGAAGAACTC-30
129
AF179871
[27]
Please cite this article in press as: Marangi M, et al., Multiplex PCR for the detection and quantification of key zoonotic genotypes of Giardia, Cryptosporidium and Toxoplasma in wastewater and mussels, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.01.001
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calculated using the formula: CV ¼ s(Ct)/m(Ct), as described in the manufacturer's protocol (BioRad). 3. Results and discussion First, we assessed the performance of the qPCR assay. An amplification efficiency (E) of 100.7% was achieved, with a slope of 3305 (R2 ¼ 0.999). The linearity of amplification from 1010 to 10 copies per ml was regarded as acceptable, and a minimum of 10 gene copies was detected for each of the three target protists. Intraand inter-assay variabilities were estimated at 1.6e1.7% and 0.90e0.95%, respectively. We then applied the assay to test 232 wastewater and mussel DNA samples (Table 2). In total, 34 of the 119 (28.6%) water samples were test-positive for either C. parvum or G. duodenalis or both protists; 10 (8.4%) of all 119 samples were test-positive for G. duodenalis, 18 (15.1%) for C. parvum, and 6 (5%) for G. duodenalis and C. parvum. All 119 samples were test-negative for T. gondii. In test-positive samples, the numbers of oocysts/cysts were estimated at 10 to 56 for C. parvum and 7 to 118 for G. duodenalis. Subsequently, we tested the mussel DNA samples. Seven of the 53 (13.2%) mussel DNA samples from Turkey were test-positive for T. gondii. All of these samples were test-negative for G. duodenalis and C. parvum. In addition, 40 of the 60 (66.6%) samples from Italy were test-positive for one or more target protists. Specifically, 26 (43.3%) of the 60 samples were test-positive for C. parvum, six (10%) for G. duodenalis and 8 (13.3%) for both protists; all 113 samples were test-negative for T. gondii. For test-positive mussel samples, the numbers of oocysts/cysts were estimated at 10 to 64 for C. parvum, 4 to 78 for G. duodenalis (Italy) and 6 to 30 for T. gondii (Turkey) per 5 ml of genomic DNA tested. Finally, the sequencing of wastewaterand mussel-DNA derived amplicons confirmed the specific identity of G. duodenalis assemblage A (n ¼ 24), C. parvum (n ¼ 36) and T. gondii Type I (n ¼ 7). This multiplex qPCR for the simultaneous detection of G. duodenalis (assemblage A), C. parvum and T. gondii (Type I) shows promise for use in epidemiological surveys and could assist in the monitoring and tracking disease linked to food and waterborne outbreaks. In the last decade, numerous molecular diagnostic assays for viruses and bacteria have been developed for use in diagnostic laboratories, and are now being increasingly established for a range of enteric protozoa [12,33]. Although many qPCR techniques have been developed [34,35], some of them have limitations, in terms of the cost and performance of particular dyes in
Table 2 The qPCR results for water and mussel samples tested in this study. Protists
qPCR results Number of test-positive samples/total number of samples tested Water (Italy)
Mussels (Italy) Mussels Total (Turkey) numbers (%)
Giardia duodenalis 10/119 (8.4%) 6/60 (10%) Cryptosporidium 18/119 (15.1%) 26/60 (43.3%) parvum Toxoplasma gondii 0 0 G. duodenalis & C. parvum G. duodenalis & T. gondii C. parvum & T. gondii Totals (%)
0 0
16/179 (8.9%) 44/179 (24.5%) 7/53 (13.2%) 14/179 (7.8%)
6/119 (5%)
8/60 (13.3%)
7/53 (13.2%) 0
0
0
0
0
0
0
0
0
7/53 (13.2%)
81/232 (34.9%)
34/119 (28.6%) 40/60 (66.6%)
3
PCR. For instance TaqMan probes [36] are expensive [37] or dyes, such as SyberGreen® may inhibit PCR [38], which reduces reproducibility and/or analytical sensitivity. In contrast, EvaGreen® seems to be more suitable for routine multiplex qPCR applications, as it has less inhibitory effect and lacks dye-redistribution [39,40]. The assay established here is inexpensive (costs approximately $1.50 per sample), takes 1.3 h to perform, and is quantitative and sensitive. Current evidence obtained by direct sequencing of a total of 67 amplicons indicates that the present assay should achieve specific amplification from G. duodenalis assemblage A (n ¼ 24), C. parvum (n ¼ 36) and T. gondii Type I (n ¼ 7) from wastewater and mussel samples with potential for transmission to humans, provided that cysts/oocysts of these pathogens in such samples are viable and infective. However, given the nature and extent of the different genomic DNAs that might occur in these two types of biological matrices (i.e. wastewater and mussel e a filter-feeder), such as those potentially derived from an array of viruses, bacteria, fungi, protists and/or other microorganisms or organisms (whose genomes might not yet have been sequenced), it will be crucial to keep monitoring the specificity of the qPCR (and its primers and conditions) by direct sequencing or mutation scanning [41] of amplicons produced using the three primer sets employed herein. Although melting profiles provide an indication of specificity, the qualitative analysis of amplicons by electrophoretic mutation scanning or sequencing will continue to be critical during the deployment of the present qPCR assay. This assay also could be a useful tool for the simultaneous detection and quantitation of the three zoonotic protists in various other biological matrices (such as soil, vegetables and/or fruits), but its extension to such matrices (also representing samples from different geographical origins) would require continual verification of assay specificity and sensitivity. Acknowledgements This work was partly supported by a grant from PON Puglia ERDF 2007e2013, Axis I, Line 1.2. Accordo di Programma Quadro. Reti di Laboratori Pubblici di Ricerca. Progetto L.A.I.F.F. (codice n. 47), Italy, and by a grant from PON REC 01_01480 IN.TE.R.R.A. (Technology and process innovations for irrigation reuse of treated municipal and agro-industrial wastewaters to achieve sustainable water resources management), Bari, Italy. The authors thank Dr Tiziana Caradonna for technical assistance. RBG's research was supported by the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC) of Australia, Melbourne Water Corporation, and Yourgene Bioscience; it was also supported by a Victorian Life Sciences Computation Initiative (VLSCI; grant number VR0007) on its Peak Computing Facility at the University of Melbourne, an initiative of the Victorian Government. References [1] Karanis P, Kourenti C, Smith H. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J Water Health 2007;5:1e38. [2] Dorny P, Praet N, Deckers N, Gabriel S. Emerging food-borne parasites. Vet Parasitol 2009;163:196e206. [3] Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev 2013;26:115e34. [4] Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011;24:110e40. S. Zoonotic potential of Giardia. Int J Parasitol 2013;43:943e56. [5] Ryan U, Caccio [6] Barratt JL, Harkness J, Marriott D, Ellis JT, Stark D. Importance of nonenteric protozoan infections in immunocompromised people. Clin Microbiol Rev 2010;23:795e836.
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