A novel multiplex PCR coupled with Luminex assay for the simultaneous detection of Cryptosporidium spp., Cryptosporidium parvum and Giardia duodenalis

Veterinary Parasitology 173 (2010) 11–18

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A novel multiplex PCR coupled with Luminex assay for the simultaneous detection of Cryptosporidium spp., Cryptosporidium parvum and Giardia duodenalis Wei Li a , Nan Zhang b , Pengtao Gong a , Lili Cao a , Jianhua Li a,∗ , Libo Su a , Shuhong Li c , Yumei Diao a , Kang Wu a , He Li a , Xichen Zhang a,∗ a b c

College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xian Road, Changchun 130062, China College of Life Science, Jilin University, 2699 Qianjin Street, Changchun 130012, China Basic Medical Science School, Jilin University, 828 Xinmin Street, Changchun 130021, China

a r t i c l e

i n f o

Article history: Received 5 February 2010 Received in revised form 7 April 2010 Accepted 31 May 2010 Keywords: Multiplex PCR Luminex hybridization Microsphere Cryptosporidium spp. Cryptosporidium parvum Giardia duodenalis

a b s t r a c t Cryptosporidium parvum and Giardia duodenalis are the most frequently identified enteric parasites associated with diarrhea-causing disease outbreaks, and many non-parvum species of Cryptosporidium also can replicate and cause illness in mammals including humans. In this study, we describe a novel multiplex PCR coupled with Luminex assay for the identification of Cryptosporidium spp., C. parvum and G. duodenalis in a rapid manner. The multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was developed using three pairs of biotinylated primers which amplify 424, 223 and 267 bp products from the U1 small nuclear ribonucleoprotein (U1 snr) gene, 18S rRNA gene of Cryptosporidium and the beta-giardin gene of Giardia, respectively. The genus and speciesspecific capture probes linked to carboxylated Luminex microspheres hybridized to the multiplex PCR amplicons to enhance sensitivity and specificity. The conditions of multiplex PCR and Luminex hybridization reaction were optimized to enable the minimum detection limits of 5 × 10−6 , 5 × 10−6 , and 5 × 10−6 ng DNAs (corresponding approximately to 0.1 oocyst/cyst). The Luminex approach proved to be 100% specific and accurate by testing a total of 240 fecal samples compared with microscopic examination of fecal smears and further modified acid-fast staining or iodine-staining observation. The established assay offers the potential for rapid detection of Cryptosporidium spp., C. parvum and G. duodenalis in fecal and environmental samples. © 2010 Elsevier B.V. All rights reserved.

1. Introduction In recent years, many outbreaks of cryptosporidiosis and giardiasis caused by Cryptosporidium and Giardia have been well recorded worldwide, Cryptosporidium parvum and Giardia duodenalis are the main causative agents which

∗ Corresponding authors. Tel.: +86 431 87981351; fax: +86 431 87981351. E-mail addresses: [email protected] (J. Li), [email protected] (X. Zhang). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.05.024

cause diarrhea issues for the public health (Fayer, 2004; Karanis et al., 2007). In immunocompetent hosts, they usually cause self-limiting diarrheal illness that resolve spontaneously. However, young children with nutritional deficiencies and immunocompromised hosts, especially AIDS patients, may suffer from severe, chronic and lifethreatening infection (Hunter and Nichols, 2002). The Cryptosporidium oocysts and Giardia cysts are both environmentally resistant, oral–fecal route is the main pathway through which people get infected (Karanis et al., 2007; Gonc¸alves et al., 2006). Among the valid species of Cryptosporidium (protozoa, apicomplexa), C. parvum is the most

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Table 1 All the primer pairs used for the establishment of multiplex PCR assay and the genotyping of Cryptosporidium and Giardia isolates. Primer name

Primer sequence

CF1 CR1 CF2 CR2 CF3 CR3 GF1 GR1 GF2 GR2 GF3 GR3 HF HR

5 -ATTGGAGGTTGTTCCTTACTCCT-3 5 -Biotin-CACCACCCATAGAATCAAGAA-3 5 -CTGGTTGATCCTGCCAGTAG-3 5 -TAAGGTGCTGAAGGAGTAAGG-3 5 -CAGTTATAGTTTACTTGATAATC-3 5 -CAATACCCTACCGTCTAAAG-3 5 -TACGCTCACCCAGACGATG-3 5 -Biotin-TCGGCGGACTTCTTGACCT-3 5 -TGTGGCTAAGCGTGTTGTA-3 5 -GGTGCGTTCGATGTAAGAC-3 5 -AGACCACGCTCAGTTGTTA-3 5 -TTCGTTATCCTTTCATTTGT-3 5 -Biotin-TAAGAAGCCACCAAGAAGGCG-3 5 -TCAATAGGCTTAAATGGGTTCGGGA-3

a b

Step and conditions (◦ C/s)a

No. of cyclesb

D, 94/30; A, 59/40; E, 72/30

30

D, 94/30; A, 60/45; E, 72/90

39

D, 94/30; A, 60/45; E, 72/90

39

D, 94/30; A, 59/40; E, 72/30

30

D, 94/60; A, 53/60; E, 72/60

40

D, 94/60; A, 52/60; E, 72/60

40

D, 94/30; A, 59/40; E, 72/30

30

Actual denaturation (D), annealing (A), and extension (E) temperatures and times used in this study and cited publications. Number of cycles used in this study and original publications.

prevalent zoonotic species that causes intestinal disease in humans and animals. Many other non-parvum species including Cryptosporidium hominis, Cryptosporidium felis, Cryptosporidium canis, Cryptosporidium meleagridis and Cryptosporidium muris were also reported to be associated with infection in mammals (Gatei et al., 2003; Katsumata et al., 2000; Morgan et al., 1999, 2000; Pedraza-Díaz et al., 2000, 2001; Pieniazek et al., 1999). To date, no effective vaccines and drugs have been developed for this ubiquitous parasite, making cryptosporidiosis a major public health issue and economic problem (Hoxie et al., 1997). G. duodenalis (synonym of Giardia intestinalis and Giardia lamblia) is also an intestinal protozoan found in a wide range of mammalian hosts including humans. Assemblages A and B are the two major groups associated with infection in humans, which have been isolated from the feces of cats, dogs and many livestock animals (Thompson et al., 2000). In China, there are no matching records on the large-scale outbreaks of these two diarrheal diseases yet. However, the hidden dangers exist in many aspects of daily life and may cause miscellaneous water- and food-borne infections. For many years, microscopic examination of fecal samples has been commonly considered to be the “gold standard” for diagnosis of G. duodenalis and C. parvum infections (Alles et al., 1995; Martín Sánchez et al., 1993). More specific and sensitive alternative methods (enzyme-linked immunosorbent assay and direct fluorescent-antibody assay) have also been extensively developed over the years (Arrowood and Sterling, 1989; Garcia et al., 1987; Rusnak et al., 1989; Mekaru et al., 2007). However, the assays mentioned above need to harvest enough oocysts and cysts from feces for detection, sometimes are labor-intensive, time-consuming and less sensitive, and the antibodies utilized in the direct fluorescent-antibody methods were often shown to have cross-reaction with various species of algae in environmental samples (Rodgers et al., 1995). Thus, more sensitive and specific assays are required to detect low-level contamination of oocysts and cysts. Molecular detection assays based on regular PCR, PCR/Southern blot, Real-Time PCR and Nested PCR-RFLP have the potential of addressing many of the limitations of

traditional methods and are frequently introduced in many of previous studies for genus or species-specific detection (Rochelle et al., 1997; Laberge et al., 1996; Bruijnesteijn van Coppenraet et al., 2009; Coupe et al., 2005). The advantages of PCR include greater sensitivity, relatively low cost, rapid detection of multiple pathogens, and the ability to discriminate between species and strains if suitable primers are selected. For further genotyping distinction, DNA sequencing analysis was regularly conducted (Xiao and Fayer, 2008). However, sequencing is also costly, labor-intensive, time-consuming, and needs a lab assistant to operate the DNA sequencer and do data analysis, which makes it less adequate for a rapid diagnostic response. The newly emerging Luminex detection assay based on xMAP technology (multi-analyte profiling beads) can meet the needs for rapid diagnosis and enable the detection and quantitation of multiple DNA targets. When coupled with multiplex PCR, the assay has the capacity to identify multiple infectious agents in the same reaction well at the same time (Dunbar, 2006; Taylor et al., 2001). In the present study, we have demonstrated a multiplex-Luminex assay for the simultaneous identification of Cryptosporidium spp., C. parvum and G. duodenalis. The assay was evaluated by testing a total of 240 fecal samples, and the results were compared with traditional microscopic examination and modified acid-fast staining or iodine-staining assay. 2. Materials and methods 2.1. Parasite DNAs for establishing protocols C. parvum and Cryptosporidium andersoni oocysts were isolated from feces of naturally infected cattle and goats in Changchun suburban area of China. Cryptosporidium suis and Cryptosporidium baileyi oocysts were isolated from feces of naturally infected pigs and chicken in Shanghai suburban area of China. G. duodenalis cysts were obtained from feces of cage-reared dogs in Shenyang, China. G. duodenalis trophozoites (strain CH-C2) presented by Professor Siqi Lu of China Capital Medical University, were isolated from feces of diarrhea patient in Beijing, China. The DNAs of Trichomonas vaginalis, Eimeria tenella, Eimeria nieschulzi

W. Li et al. / Veterinary Parasitology 173 (2010) 11–18

and Toxoplasma gondii as control strains were stored in our laboratory (Molecular Parasitology Laboratory of Jilin University). ZnSO4 flotation method was primarily deployed to isolate Cryptosporidium oocysts and Giardia cysts from fecal samples. The concentrated oocysts and cysts were further purified using discontinuous Sheather’s gradient centrifugation and one-layer percoll-sucrose centrifugation as previously described (Coupe et al., 2005; Mayer and Palmer, 1996). The purified oocysts and cysts were counted with a hemacytometer, diluted to standard concentrations and kept at 4 ◦ C until use. Genotypes of isolated Cryptosporidium were determined using nested PCR and DNA sequencing analysis with the primer sets of CF2 /CR2 and CR3 /CR3 (Table 1) described by Coupe (Coupe et al., 2005). Genotypes of G. duodenalis isolates obtained from caged dogs were identified using regular PCR and DNA sequencing analysis with the assemblage-specific primer sets of GF2 /GR2 and GF3 /GR3 (Table 1) which amplify 256 and 164 bp products from the hypervariable region of triosephosphate isomerase gene (GenBank accession No. L02120 and L02116). The genotyped isolates including C. parvum cattle genotype, goat genotype, C. andersoni, C. suis, C. baileyi, G. duodenalis assemblage A, assemblage B and G. duodenalis (strain CH-C2) were utilized to set up multiplexLuminex assay protocols. 2.2. Fecal samples and DNA extraction A total of 240 fecal samples were obtained from dogs (130), cattle (50), pigs (30) and goats (30) in Changchun city and Shenyang city of China. The preliminary purification was performed for all samples using ZnSO4 flotation method. The surface layer of ZnSO4 flotation solution was sandwiched between a glass plate and a cover plate for microscopic examination. The remaining surface solution was transferred into a fresh centrifuge tube, filled with PBS and centrifuged at 2500 × g for 15 min. The pellets were resuspended in 1 ml of lysis buffer (4 M urea, 200 mM Tris, 20 mM NaCl, 200 mM EDTA, pH 7.4) and 7 ␮l of proteinase K (40 mg/ml). Mixtures were then exposed to three cycles of freezing in liquid nitrogen for 5 min and thawing at 65 ◦ C for 5 min and finally incubated for 2 h at 65 ◦ C to release nucleic acids. Total nucleic acids were extracted by phenol extraction and isopropanol precipitation in the presence of 1 M ammonium acetate, dissolved in TE water and used as templates in PCR. 2.3. Multiplex-Luminex assay 2.3.1. Multiplex PCR 2.3.1.1. Primer design for multiplex PCR. For Cryptosporidium-specific detection, various available 18S rRNA sequences of Cryptosporidium deposited in GenBank were downloaded and aligned using DNAMAN Software (version 6.0, Lynnon Biosoft Corporation, QC, Canada). After the detailed alignment, the genus-specific primer pair of CF1 /CR1 which amplifies a 223-bp fragment targeting the conservative region of 18S rRNA gene was devised. For C. parvum-specific detection, the primer set of HF/HR which amplifies a 424-bp fragment targeting

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the U1 snr gene (GenBank accession No. XM 626719) was designed. For G. duodenalis-specific detection, the primer pair of GF1 /GR1 which amplifies a 267-bp fragment targeting the beta-giardin gene (GenBank accession No. X85958) was devised. All primer sequences were analyzed using Oligo Primer Analysis Software (version 6.0, National Biomedical Systems, USA) and facilitated by application of the BLAST program for the specificity prediction (www.blast.ncbi.nlm.nih.gov/Blast.cgi). 2.3.1.2. Optimization of multiplex PCR conditions. To confirm that the individual primer pairs were eligible for the amplification of all three gene fragments, the single-target PCR conditions including primer concentration, annealing temperature, dNTP concentration, MgCl2 concentration, and amount of DNA polymerase were first optimized. The optimal conditions appropriate for all three individual primers were elected for multiplex PCR amplification. In order to amplify all of the specific PCR products with equal efficiency, an optimization strategy of multiple primers was described as follows. Duplex PCR was performed by mixing two individual primer sets of HF/HR and CF1 /CR1 in different ratios (0.5/0.4 ␮M, 0.6/0.4 ␮M, 0.7/0.4 ␮M, 0.8/0.4 ␮M, 0.9/0.5 ␮M and 1/0.5 ␮M) for primer concentration test, triplex PCR was conducted by mixing three individual primer sets of HF/HR, GF1 /GR1 and CF1 /CR1 in different ratios (0.5/0.5/0.5 ␮M, 0.6/0.5/0.4 ␮M, 0.7/0.4/0.4 ␮M, 0.7/0.4/0.3 ␮M, 0.8/0.3/0.4 ␮M and 0.8/0.3/0.3 ␮M) for primer concentration test. The cycle numbers of multiplex PCR and the duration for denaturation, annealing and elongation were also experimentally tested. 2.3.1.3. Multiplex PCR amplification. After the optimization, a uniform set of conditions were selected to perform multiplex PCR amplification. The reaction mix for multiplex PCR in a final volume of 50 ␮l consisted of 10 mM Tris–HCl, 1.5 mM MgCl2 , 50 mM KCl, 250 ␮M dNTPs, 0.7/0.4/0.4 ␮M primer pairs, 1% dimethyl sulfoxide, DNA templates corresponding to 104 oocysts of Cryptosporidium isolates, 104 cysts of G. duodenalis isolates, 105 trophozoites of G. duodenalis (CH-C2) and 8 ng of other control stains for specificity tests and DNA templates (C. parvum cattle genotype and G. duodenalis strain CH-C2) diluted into 1 × 10−2 , 5 × 10−3 , 1 × 10−3 , 5 × 10−4 , 1 × 10−4 , 5 × 10−5 and 1 × 10−5 ng for sensitivity tests. The mixtures were added with 2 U Ex Taq DNA polymerase (TaKaRa, Japan) and 0.05 U UNG (Uracil-DNA Glycosylase, Fermentas, USA), then put in the T-personal thermal cycler (Biometra, Germany). After being held at 50 ◦ C for 2 min to active UNG to prevent potential carry-over contamination from previous amplifications, all prepared PCR mixtures were then denatured at 94 ◦ C for 30 s, annealed at 59 ◦ C for 40 s, and extended at 72 ◦ C for 30 s. This cycle was repeated 30 times, and the samples underwent a final elongation at 72 ◦ C for 7 min. The amplified fragments were separated by electrophoresis in a 1.5% agarose gel in Tris–acetate buffer (0.04 M Tris–acetate, 0.001 M EDTA [pH 8.0]) for 40 min at 80 V, stained with ethidium bromide (0.2 mg/ml), and photographed on a UV transilluminator. Cloning and sequencing were also performed to validate the reliability of multiplex PCR. Briefly,

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the distinct DNA bands appeared in agarose gels were respectively excised and purified, then cloned into pMDTM 18-T Vector (TaKaRa, Japan). After clone screening, the positive clones were selected and sequenced for two strands. 2.3.2. Luminex hybridization 2.3.2.1. Probe design. To design the specific capture probes for Luminex hybridization reaction, the 18S rRNA gene, U1 snr gene of Cryptosporidium and beta-giardin gene of Giardia respectively deposited in GenBank under accession numbers EU675852, XM 626719 and X85958 were utilized. These sequences were downloaded and analyzed using Oligo Primer Analysis Software (version 6.0, National Biomedical Systems, USA). Based on the probe design guidelines of Luminex® 200TM analyzer (Luminex Corp., Austin, TX, USA), the successfully designed Cryptosporidium-specific probe (ProC) was 5 ACTCACCAGGTCCAGACATAG-3 , the C. parvum-specific probe (ProH) was 5 -CTTTCGGACCTCTTCCTCATC-3 and the G. duodenalis-specific probe (ProG) was 5 CTCAGCAACATGAACCAGCG-3 . The selected probe sequences were amino modified at the 5 end and attached to a 12-carbon linker previously determined to be functionally optimal for microsphere-based hybridization assays (Cowan et al., 2004). 2.3.2.2. Optimization of hybridization conditions. Two DNA samples extracted from C. parvum (cattle genotype) and G. duodenalis (strain CH-C2) were used for optimization experiments, each optimization experiment was performed at least twice with duplicate data points. For hybridization temperature, five different hybridization temperatures (37 ◦ C, 42 ◦ C, 47 ◦ C, 52 ◦ C and 57 ◦ C) were tested, hybridization time was 30 min and the concentration of streptavidin-R-phycoerythin was 2 ␮g/␮l diluted in 1× TMAC buffer. For hybridization time, various hybridization times (15 min, 30 min, 45 min and 60 min) were tested with a hybridization temperature of 52 ◦ C and streptavidinR-phycoerythin dilution of 2 ␮g/␮l. 2.3.2.3. Luminex-based hybridization. A carbodiimide coupling procedure was used to couple the three capture probes to 5.6-␮m, polystyrene, carboxylated microspheres (Luminex Corp., Austin, TX, USA). The hybridization reaction was based on the binding of the complementary capture probes to the biotinylated PCR products using the procedure described previously (Bandyopadhyay et al., 2007). The Luminex® 200TM platform (Luminex, USA) processes signal for each of the two different fluorophores excited by two lasers with different wavelengths which respectively detect the internal dyes of the addressed microspheres and the R-phycoerythrin conjugated to capture probes. For data acquisition, the median fluorescence intensity (MFI) of the streptavidin-R-phycoerythin conjugate bound to 3 of each microsphere population was calculated using Luminex digital signal processors and proprietary software. 2.3.2.4. Result interpretation. The presence or absence of the Cryptosporidium or Giardia species was evaluated by the value of the signal/blank ratio (S/B ratio) of MFIs, which

were calculated and designated as the signal ratio (SR). Signals were generated only when biotinylated sequences bound to the complementary probe on the respective microsphere population. Positive sample indicated an SR of ≥2 and negative sample indicated an SR of <2. According to the test results, if C. parvum oocysts were present in the sample, the SR values of ≥2 were obtained both from the ProH and ProC populations, if non-parvum oocysts of Cryptosporidium were present in the sample, the SR value of ≥2 was obtained only from ProC population, and if G. duodenalis cysts were present in the sample, the SR value of ≥2 was obtained only from ProG population. The negative samples had the lowest MFIs, similar to the blanks. 2.3.2.5. Specificity and sensitivity of Luminex assay. To determine the specificity, the DNA was extracted from large numbers of oocysts and cysts. Quantities of DNA corresponding to 104 purified Cryptosporidium oocysts and Giardia cysts and 105 in vitro cultured trophozoites of G. duodenalis (CH-C2) were adopted. DNAs of T. vaginalis, E. tenella, E. nieschulzi and T. gondii were used at a final concentration of 8 ng. A series of dilutions of C. parvum (cattle genotype) and G. duodenalis (strain CH-C2) DNAs including 1 × 10−2 , 5 × 10−3 , 1 × 10−3 , 5 × 10−4 , 1 × 10−4 , 5 × 10−5 , 1 × 10−5 , 5 × 10−6 and 1 × 10−6 ng were deployed to validate the sensitivity of Luminex assay. 2.4. Detection of fecal samples To validate the applicability and accuracy, we further applied our multiplex-Luminex assay to detect 240 fecal samples mentioned above. Microscopic observations combined with modified acid-fast staining and iodine-staining assay were primarily performed to identify Cryptosporidium oocysts and Giardia cysts, respectively. The fecal samples which were detected as positive by Luminex assay but negative by microscopic examination and staining method were further confirmed by nested/regular PCR followed by DNA sequencing using the species-specific primer pairs (CF2 /CR2 , CF3 /CR3 and GF1 /GR1 ) (Table 1). 3. Results 3.1. Microscopic examination Based on the experimental results of microscope observation and staining technique, 30 (Giardia), 10 (Cryptosporidium), 3 (Cryptosporidium) and 2 (Cryptosporidium) positive samples respectively obtained from dogs (total 130), cattle (total 50), pigs (total 30) and goats (total 30) were ultimately determined (Table 2). 3.2. Optimization of multiplex PCR conditions For duplex PCR amplification, the concentrations of 0.5/0.5 ␮M were tested to be optimal, which could amplify two distinct bands with equal efficiency (Fig. 1a). For triplex PCR amplification, three distinct bands corresponding to their respective molecular sizes were amplified with the

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Fig. 1. The establishment of multiplex PCR assay. Lane 1 of panels a, b and e and Lane 9 of panels c and d = DL2000 DNA ladders (TaKaRa, Japan). Panels a and b represent the optimization of duplex and triplex primer concentration. Panels c, d and e respectively represent the sensitivity and specificity tests of multiplex PCR. Panels c and d demonstrate the gradient detection with serial template dilutions including 1 × 10−2 , 5 × 10−3 1 × 10−3 , 5 × 10−4 , 1 × 10−4 , 5 × 10−5 , 1 × 10−5 ng DNAs and negative control. Lanes 2–13 of panel e: strain controls of C. parvum cattle genotype, goat genotype, C. andersoni, C. suis, C. baileyi, G. duodenalis assemblage A, assemblage B, T. vaginalis, E. tenella, T. gondii, E. nieschulzi and negative control.

concentrations of 0.7/0.4/0.4 ␮M (Fig. 1b). Multiple amplifications with cycle numbers of 30 times, 35 times and 40 times, appeared to be no notable distinction. The duration of 30 s for denaturation, 40 s for annealing and 30 s for elongation was tested to be optimal and finally adopted.

the highest MFIs for three capture probes. The optimal hybridization time proved to be 30 min as the reduced signals were observed at both higher and lower hybridization times.

3.3. Optimization of hybridization conditions

3.4. Specificity and sensitivity of multiplex-Luminex assay

The hybridization temperature of 52 ◦ C was found to be optimal for hybridization reaction, which generated

Prior to Luminex hybridization reaction, the primarily performed multiple amplifications exhibited favorable

Table 2 Detection of fecal samples using Luminex assay compared with smear and staining assay. Animals

Smear and stain assay (+/Total)a Luminex assay (+/Total)b Confirmationc a b c

Dog

Cattle

Pig

Goat

30/130 32/130 2(+)

10/50 11/50 1(+)

3/30 6/30 3(+)

2/30 5/30 3(+)

Microscopic examination of fecal smears and further modified acid-fast staining or iodine-staining observation. Multiplex PCR coupled with Luminex assay. Confirming the negative samples for smear and staining assay but positive for Luminex assay by nested/regular PCR combined with DNA sequencing.

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Fig. 2. The specificity and sensitivity of Luminex assay. Panel a indicates the specificity of Luminex assay by testing TE control, negative control and a series of strain controls including C. parvum cattle genotype, goat genotype, C. andersoni, C. suis, C. baileyi, G. duodenalis assemblage A, assemblage B, T. vaginalis, E. tenella, T. gondii, and E. nieschulzi. Panel b indicates the sensitivity of Luminex assay by testing serial dilutions of DNA templates containing 1 × 10−2 , 5 × 10−3 , 1 × 10−3 , 5 × 10−4 , 1 × 10−4 , 5 × 10−5 , 1 × 10−5 , 5 × 10−6 , 1 × 10−6 ng and negative control.

specificity, which produced distinct bands corresponding to their respective molecular sizes. No cross- and unspecific-amplification was recognizable in agarose gels (Fig. 1e). The cloned sequences were shown to be 100% corresponding to the GenBank database for sequence homology. The Luminex hybridization also showed favorable specificity, which was quite compatible with the results produced by multiplex PCR (Fig. 2a). With regard to the sensitivity, 1 × 10−4 and 5 × 10−5 ng DNAs were simultaneously detected by duplex PCR (Fig. 1c). 1 × 10−4 , 5 × 10−5 and 5 × 10−5 ng DNAs were simultaneously detected by triplex PCR (Fig. 1d). The results were quite compatible with the detection level of single-target PCR (data not shown). When coupled with Luminex assay, the sensitivity had a notable enhancement with the simultaneous detection limits of 5 × 10−6 , 5 × 10−6 and 5 × 10−6 ng DNAs, which were 20-fold, 10fold and 10-fold higher than that only detected by multiplex PCR (Fig. 2b). In addition, 5 × 10−5 ng DNA is corresponding approximately to 1 oocyst/cyst (Coupe et al., 2005). 3.5. Detection of fecal samples Four samples detected as positive by Luminex assay but as negative by “gold standard” were further identified to be C. parvum (goat), C. suis (pig), C. andersoni (cattle) and G. duodenalis (dog) by PCR assay coupled with DNA sequencing described previously. All negative samples identified by Luminex assay probed to be 100% corresponding to that identified by the “gold standard” (Table 2). 4. Discussion Most of the previous PCR detection assays were specific and sensitive only for one particular infectious agent, and subsequent experiments needed to be performed to confirm its species or genotype (Mahbubani et al., 1992; Bertrand et al., 2005). There were very few reports on multiplex PCR and DNA-based Luminex applications for the detection of protozoons. A recent study demonstrated the

usefulness of this technique for multiplex SNP discrimination between C. parvum and C. hominis (Bandyopadhyay et al., 2007). Several other studies based on Luminex platform had also been developed to detect infectious agents including Plasmodium, Escherichia coli, Listeria and Mycobacterium using direct or competitive DNA hybridization assay, which could identify multiple infectious agents or different genotypes of one infectious agent (Carnevale et al., 2007; McNamara et al., 2006; Cowan et al., 2004; Das et al., 2006; Diaz and Fell, 2004; Dunbar et al., 2003). In our study, a novel, robust and rapid multiplexLuminex assay was proposed to simultaneously detect Cryptosporidium spp., C. parvum and G. duodenalis, with potential applications in environmental surveillance and human diagnostics. To the best of our knowledge, our study represents the first multiple detection of Cryptosporidium and Giardia using Luminex assay, which can simply be used as a tool to evaluate the fecal samples for large amounts at one time and meet the requirements for nice sensitivity and for fast detection within 5 h. However, this assay has a limitation that it cannot discriminate between non-parvum species of Cryptosporidium and assemblages of G. duodenalis as the design of primers and probes is based on the needs for wide spectrum detection. 18S rRNA gene is the most widely characterized and described sequence compared to other gene sequences (COPW, HSP70 and undefined genes) for Cryptosporidium PCR identification. This gene has sequence similarity between different species of Cryptosporidium, which enables the excellent design of genus-specific primers and probes. Besides the wide spectrum detection of Cryptosporidium, the U1 snr gene was potentially applied to devise species-specific primers and probes to discriminate C. parvum from other closely related species, because this gene deposited in GenBank is uniquely described for C. parvum, no U1 snr sequences of other Cryptosporidium species have been deposited thus far in GenBank. Several polymorphic regions were shown in beta-giardin gene of Giardia by multiple sequence alignment over the whole sequence length described in previous study (Mahbubani et al., 1992). Thus, the beta-giardin gene has the potential of

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designing primers and probes to discriminate G. duodenalis from G. muris and G. ardeae. The triosephosphate isomerase (TPI) gene of Giardia was also widely deployed as diagnostic sequence, however it is commonly used for identification of various assemblages of G. duodenalis (Bertrand et al., 2005). High DNA hybridization signal ratio (SR) mainly depends on the excellent design of primers and probes, and on the use of optimized PCR and hybridization conditions. In our study, the tested primers (CF1 /CR1 , HF/HR and GF1 /GR1 ) had the ideal combination of sensitivity, specificity and compatibility with multiplex PCR, and the probes (ProC, ProH and ProG) covalently linked to different addressed microspheres could lead to high SR in Luminex hybridization reactions. Other critical factors influencing SR include the primer concentration of multiplex PCR and the hybridization temperature and time. The primer concentration of multiplex PCR needs to be optimized to avoid primer dimmers and non-specific products which can interfere with the amplification of specific products, and the hybridization temperature and time were also required to be optimized to generate high hybridization efficiency. In conclusion, multiplex PCR is supposed to become a more widely accepted assay for the detection of parasitic protozoon and should be coupled with Luminex assay to improve detection capabilities. Acknowledgements This study was supported by the National Key Technology R&D Program of China (No. 2007BAD40B05) and High Technology Research and Development Program (863) of China (No. 2006AA10A207). We take many thanks to supervisors and others who provided laboratory assistance and helpful suggestions and advice. We declare that the experiments comply with the current laws of China where they were performed. References Alles, A.J., Waldron, M.A., Sierra, L.S., Mattia, A.R., 1995. Prospective comparison of direct immunofluorescence and conventional staining methods for detection of Giardia and Cryptosporidium spp. in human fecal specimens. J. Clin. Microbiol. 33, 1632– 1634. Arrowood, M.J., Sterling, C.R., 1989. Comparison of conventional staining methods and monoclonal antibody-based methods for Cryptosporidium oocyst detection. J. Clin. Microbiol. 27, 1490–1495. Bandyopadhyay, K., Kellar, K.L., Moura, I., Casaqui Carollo, M.C., Graczyk, T.K., Slemenda, S., Johnston, S.P., da Silva, A.J., 2007. Rapid microsphere assay for identification of Cryptosporidium hominis and Cryptosporidium parvum in stool and environmental samples. J. Clin. Microbiol. 45, 2835–2840. Bertrand, I., Albertini, L., Schwartzbrod, J., 2005. Comparison of two target genes for detection and genotyping of Giardia lamblia in human feces by PCR and PCR-restriction fragment length polymorphism. J. Clin. Microbiol. 43, 5940–5944. Bruijnesteijn van Coppenraet, L.E., Wallinga, J.A., Ruijs, G.J., Bruins, M.J., Verweij, J.J., 2009. Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy. Clin. Microbiol. Infect. 15, 869–874. Carnevale, E.P., Kouri, D., DaRe, J.T., McNamara, D.T., Mueller, I., Zimmerman, P.A., 2007. A multiplex ligase detection reaction-fluorescent microsphere assay for simultaneous detection of single nucleotide polymorphisms associated with Plasmodium falciparum drug resistance. J. Clin. Microbiol. 45, 752–761. Coupe, S., Sarfati, C., Hamane, S., Derouin, F., 2005. Detection of Cryptosporidium and identification to the species level by nested PCR

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