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Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis
Journal of Microbiological Methods 37 (1999) 193–200
Journal of Microbiological Methods
Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis Ming-Qi Deng, Dean O. Cliver* Department of Population Health and Reproduction, School of Veterinary Medicine, University of California at Davis, One Shields Ave., Davis, CA 95616 -8743, USA Received 16 February 1999; received in revised form 15 April 1999; accepted 26 April 1999
Abstract A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains. 1999 Elsevier Science B.V. All rights reserved. Keywords: Giardia duodenalis; RAPD; AP-PCR; DNA extraction; Fingerprinting
1. Introduction The protozoan parasite Giardia duodenalis (synonyms: G. intestinalis or G. lamblia) is a commonly reported waterborne pathogen capable of causing prolonged gastrointestinal disease in infected humans and various mammals (LeChevallier and Norton, 1995). It occurs throughout the world in both developed countries and developing areas (Craun, 1990). However, the taxonomic criteria of G. duodenalis based on host specificity and cell *Corresponding author. Tel.: 1 1-530-754-9120; fax: 1 1-530752-5845. E-mail address: [email protected] (D.O. Cliver)
morphology have been controversial, as extensive variation in G. duodenalis has been reported from various studies. G. duodenalis strains / isolates of different origins have been characterized using methods such as isoenzyme electrophoretic analysis, antigenic comparison, restriction endonuclease analysis, and pulsed field gel electrophoresis analysis (Andrews et al., 1989; Homan et al., 1992; Nash, 1992; Nash et al., 1985; Proctor et al., 1989; Sarafis and Issac-Renton, 1993). In the past few years, polymerase-chain reaction (PCR)-based fingerprinting methods requiring smaller quantities of DNA and less parasite material have also been applied, including amplification and sequencing of a 183-bp region of the 18S rRNA gene (Weiss et al., 1992), sequence
0167-7012 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 99 )00067-6
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analysis of the 59-end of the 16S rRNA following amplification (Van Keulen et al., 1995), restriction fragment length polymorphisms (RFLP) of a glutamate dehydrogenase gene segment or other genomic region (Homan et al., 1998; Monis et al., 1996; Monis and Andrews, 1998), and randomly amplified polymorphic DNA (RAPD) analysis (McRoberts et al., 1996; Meloni et al., 1995; Morgan et al., 1993; Van Belkum et al., 1993). RAPD differs from normal PCR in that a single arbitrary primer is used instead of two sequencespecific primers. Compared to other PCR-based fingerprinting methods, RAPD requires no sequence information of a particular genomic region and there is no cloning or sequencing (Hadrys et al., 1992; Williams et al., 1993). Although RAPD is susceptible to contaminant-organism or foreign DNA, this has not been a major concern for G. duodenalis, since trophozoites from axenic cultures of G. duodenalis strains were generally used for molecular characterization. By applying the RAPD technique, it has been shown that axenic isolates of G. duodenalis from different locations could be differentiated and classified into different groups, corresponding to the classification by other techniques (McRoberts et al., 1996; Meloni et al., 1995; Morgan et al., 1993; Van Belkum et al., 1993). Our goals in this study were to establish a rapid RAPD procedure by applying quick genomic DNA preparation methods and to evaluate new arbitrary primers to facilitate molecular characterization of G. duodenalis strains.
2. Materials and methods
2.1. G. duodenalis strains Trophozoites of five human-origin G. duodenalis strains, isolated from clinical samples of various sources and cultured axenically for long terms, were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA): Portland-1 (ATCC 30888), WB (30957), KS (50114), New Orleans-1 (50137), and UNO / 04 / 87 / 1 (50184). They were grown axenically in borosilicate tubes at 378C in Keister modified Diamond’s TYI-S-33 medium as previously described (Keister, 1983). After they had grown to form a monolayer, trophozoites were
detached from the borosilicate tube surfaces by chilling in ice-water for 15 min, pelleted by centrifugation at 500 3 g for 10 min, washed once with distilled water and once with 1 3 Tris-EDTA buffer (TE, containing 10 mM Tris base and 1 mM EDTA, pH 8.0), enumerated under a hemacytometer, and resuspended in 1 3 TE to the concentration of 1.0 3 10 7 / ml. The suspension containing 1 3 10 6 trophozoites (100 ml) was then used for genomic DNA preparation as described below.
2.2. Extraction of genomic DNA Two simple methods were applied. The freezingthawing treatment was done by immersing 0.5 ml centrifuge tubes containing 100 ml of trophozoite suspension in liquid nitrogen for 2 min and then incubating in a 658C waterbath for 2 min, which was repeated five times; the supernatant following a 5min centrifugation at 10 000 3 g was stored at 2 208C. Sonication was carried out by incubating 0.5 ml tubes containing the trophozoite suspension in a waterbath-type sonicator (Branson B-52 ultrasonic cleaner, Branson Cleaning Equipment Company, Shelton, CT, USA) filled with ice-water for 15 s (short time sonication) or 5 min (long time sonication); the supernatant following a 5-min centrifugation at 10 000 3 g was stored at 2 208C. To evaluate the efficiencies of these two methods, genomic DNA was also extracted from trophozoites using a conventional DNA isolation method involving proteinase K digestion, phenol / chloroform / isoamyl alcohol extraction, and isopropanol precipitation, as was described elsewhere (Deng et al., 1997). Extracted DNA was eventually resuspended in 100 ml 1 3 TE and stored at 2 208C until further analysis.
2.3. RAPD primers A total of 10 RAPD primers were designed: GPD51 (AGC GTA CAC T), GPD52 (ATT GCG TCG A), GPD53 (GCT TGT GAA C), GPD61 (GCT TGT GAA C), GPD62 (CAA TGC CCG A), GPD63 (CAA TGC CCG A), GPD71 (TGC GCA GCT G), GPD72 (ACG AGC GTG G), GPD81 (CCA GCG TCG C), and GPD82 (GGC CGA CCG T). They are all 10 nucleotides long, with 50–80%
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G–C content, and contain no palindromic sequence. GPD62 and GPD63 had been used in an earlier study, in which both primers demonstrated their abilities in differentiating isolates of another protozoan parasite, Cryptosporidium parvum (Deng and Cliver, 1998).
2.4. Optimization of arbitrarily primed-polymerase chain reaction (AP-PCR) The PCR reaction mixture (25 ml) contained 1 3 PCR Buffer II (Perkin-Elmer Cetus Corp., Norwalk, CT, USA), MgCl 2 solution (Perkin-Elmer), each of the four deoxynucleoside triphosphates (dNTPs, Perkin-Elmer), a 10-nucleotide primer (GeneMed Biotechnologies, San Francisco, CA, USA), 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer), and template DNA suspension. It was overlaid with 25 ml of mineral oil to prevent evaporation. PCR amplification was performed on a Progene 120 Thermal Cycler (Techne Inc., Princeton, NJ, USA), using a standard RAPD thermal cycling program: after denaturation at 948C for 3 min, the reaction mixtures underwent 45 cycles of denaturation at 948C for 1 min, annealing at 368C for 1 min, and extension at 728C for 2 min, with an additional 7-min extension at 728C. AP-PCR parameters were optimized using primer GPD62, strain Portland-1, and genomic DNA extracted by freezing-thawing treatment. The MgCl 2 concentration was altered from 2.0 to 8.0 mM, primer concentration varied from 0.25 to 2.0 mM, and dNTP concentrations ranged from 50 to 400 mM. The optimal amount of template DNA in a 25 ml reaction was determined by varying the volume of extracted genomic DNA suspension and referring it to the trophozoite numbers, which ranged from 5 3 10 3 to 1.5 3 10 6 . Following AP-PCR, 20 ml of product was separated on 1.8% agarose gel, stained with 0.5 mg / ml ethidium bromide, and examined under a UV-transilluminator. A 1.0 kb DNA ladder (GIBCO BRL Life Technologies, Gaithersburg, MD, USA) was included in electrophoresis as a size marker. To ensure the RAPD reproducibility, all AP-PCR reactions were repeated three times; a successful RAPD was defined as one with multiple distinctive
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bands and with identical profiles from reactions of different batches.
2.5. Evaluation of primers and comparison of RAPD patterns of G. duodenalis strains Conditions chosen as optimum were used in all subsequent analyses for primer evaluation and comparison of RAPD patterns of the five G. duodenalis strains. Primers were evaluated as useful in the assay by two criteria: (1) they exhibited polymorphisms among different strains; and (2) the polymorphisms were reproducible among samples of the same strain. RAPD patterns were compared for the number, size, and intensity of major (distinctive) bands. Differences in intensities of minor bands were neglected.
3. Results and discussion
3.1. Optimization of AP-PCR parameters With the MgCl 2 concentrations escalating from 2.0 mM to 4.0 mM (Fig. 1, lanes 1–5), more
Fig. 1. RAPD optimization: MgCl 2 concentration. Other AP-PCR parameters (in a total of 25 ml reaction mixture) were: 200 mM of each of four dNTPs, 1.0 mM of GPD62, 2.5 U of Taq DNA polymerase, and template DNA from 5 3 10 4 trophozoites of Portland-1 strain by freezing-thawing treatment. Lane M, DNA size marker (1 kb ladder), the arrow-headed bands (from top to bottom) are approximately 2.0, 1.6, 1.0, and 0.5 kb, respectively; lanes 1–9, patterns obtained at MgCl 2 concentration 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0 mM, respectively.
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amplification products were obtained; and the intensities of the small-size bands ( , 0.8 kb) increased, while the intensities of large-size bands decreased. MgCl 2 concentrations over 4.0 mM had little further effect on the number of products; however, the intensities of larger size bands decreased (Fig. 1, lanes 6–9) and the RAPD profile became less reproducible, probably because amplification became less specific under high concentrations of Mg 11 . Therefore, the optimal concentration of MgCl 2 was determined as 4.0 mM. Similarly, the optimal concentrations of dNTP and primer were determined as 200 mM and 1.0 mM respectively. The optimal template amount for a single tube (25 ml) reaction was determined as the amount of DNA prepared from 5 3 10 4 trophozoites. As shown in Fig. 2, more distinctive bands were obtained and the intensities of the bands were higher from such a trophozoite number (lane 4). When more template was used, fewer bands with higher intensities were generated (lanes 1–3). On the other hand, using less template usually resulted in more small size bands (especially bands , 0.5 kb) with reduced intensities (lanes 5–6), and the product profile was less reproducible (data not shown). This agrees with a previous observation of RAPD reproducibility affect-
Fig. 2. RAPD optimization: template amount. Other AP-PCR parameters (in a total of 25 ml reaction mixture) were: 2.0 mM of MgCl 2 , 200 mM of each of four dNTPs, 1.0 mM of GPD62, 2.5 U of Taq DNA polymerase. Lane M, 1 kb DNA ladder; lanes 1–6, template DNA from Portland-1 strain prepared by freezing-thawing treatment from 1.5 3 10 6 , 5 3 10 5 , 1 3 10 5 , 5 3 10 4 , 1.5 3 10 4 , 5 3 10 3 trophozoites respectively.
ed by template DNA concentrations (Davin-Regli et al., 1995).
3.2. Comparison of DNA extraction methods As shown in Fig. 3, similar RAPD profiles were obtained by all three methods and they were all reproducible, indicating that methods of DNA preparation had no effect on RAPD. Although there were minor differences in RAPD profiles when DNA extracted by sonication was used, the differentiation ability of RAPD is unlikely to be affected, because only absence or presence of distinctive bands and major differences in intensities of those bands matter in comparing RAPD profiles. Although the proteinase K–phenol method usually generated bands with somewhat higher intensities (lanes 3–4), the main disadvantage of this method is the intensive hands-on manipulations and the time required for the process, which becomes tedious when a large number of samples are to be analyzed. In contrast both the freezing–thawing treatment (lanes 1–2) and sonication (either short-time or long-time, lanes 5–6 and lanes 7–8) can be completed within 30 min and there is little manipulation involved, minimizing possible
Fig. 3. Comparison of genomic DNA preparation methods. APPCR parameters (in a total of 25 ml reaction mixture) were: 2.0 mM of MgCl 2 , 200 mM of each of four dNTPs, 1.0 mM of GPD62, 2.5 U of Taq DNA polymerase, and genomic DNAs from 5 3 10 4 trophozoites of Portland-1 strain. Lane M, DNA size marker; lanes 1–2, DNA prepared by freezing-thawing treatment; lanes 3–4, DNA prepared by proteinase K–phenol method; lanes 5–6, DNA prepared by short-time (15 s) sonication; lanes 7–8, DNA prepared by long-time (5 min) sonication.
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accidental contamination of samples during the genomic DNA extraction process. Although sonication can partially degrade DNA, the insensitivity of RAPD to different DNA extraction methods observed in this study demonstrated that degraded DNA can be used in RAPD, echoing a previous report that reproducible and complex band patterns could be obtained from RAPD using partially degraded parasite (Schistosoma mansoni) DNA (Neto et al., 1997). However, our results differred from another report that different DNA extraction methods (Chelex, microwave and phenol / chloroform method) led to significantly different RAPD patterns of gram-positive bacteria Staphylococcus epidermidis and Streptococcus faecalis (Abed et al., 1995). In a subsequent investigation, genomic DNAs extracted by freezing–thawing treatment or longtime (5 min) sonication were applied to normal PCR using several sequence-specific primer pairs. Consistently, a product of the expected length - ranging from 171 bp to 1852 bp and correspondent to the specific primer pair, was obtained (data not shown here); suggesting that these simple DNA extraction methods are also effective in preparing templates for other PCR-based fingerprinting methods.
3.3. Evaluation of primers The results are shown in Fig. 4, panels A–E. Among the 10 primers investigated, GPD51 (Fig. 4A, lanes 1–5), GPD63 (Fig. 4C, lanes 6–10), and GPD71 (Fig. 4D, lanes 1–5) failed to produce polymorphisms among the G. duodenalis strains. GPD53 (Fig. 4C, lanes 1–5) and GPD62 (Fig. 4B, lanes 6–10) produced polymorphisms, but the polymorphisms were not reproducible. The remaining five primers, GPD52 (Fig. 4B, lanes 1–5), GPD61 (Fig. 4A, lanes 6–10), GPD72 (Fig. 4D, lanes 6–10), GPD81, and GPD82 (Fig. 4E) all produced reproducible polymorphisms and thus have the potential to be used in RAPD analysis for strain differentiation.
3.4. Comparison of RAPD patterns of G. duodenalis strains This was based on comparison of RAPD profiles generated from primers GPD 52, 61, 72, 81, and 82.
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Regardless of the primer used, profiles of strains Portland-1 and WB were always identical, indicating that the belong to the same group. The profiles of strains KS and UNO / 04 / 87 / 1 were also identical, with the only exception being the difference in intensities of a | 1.4 kb band generated by GDP61 (weak in strain KS, strong in UNO / 04 / 87 / 1) (Fig. 4A, lanes 8 and 10), suggesting that they are in the same group. They were readily distinguishable from profiles of strains of Portland-1 and WB, indicating that they belong to a different group. Profiles of strain New Orleans-1 and those of KS and UNO / 04 / 87 / 1 were always distinguishable, regardless the primer used in AP-PCR, indicating that New Orleans-1 should be in a different group from strains KS and UNO / 04 / 87 / 1. In contrast, the profile of New Orleans-1 and those or Portland-1 / WB had high similarities when primers GPD 72, 81, and 82 were used and were distinguishable when GPD 52 and 61 were used. Therefore, these five G. duodenalis strains seemed to belong to at least two groups: one consists of strains KS and UNO / 04 / 87 / 1, while the other consists of strains Portland-1 and WB. The New Orleans-1 strains could be in the same group as Portland-1 and WB or a third group. Although a very limited number of isolates were used in this study, apparent genetic diversity was observed even among these G. duodenalis strains, which were all from human sources. This showed the possible existence of morphologically similar but genetically distinct (cryptic) species within G. duodenalis (Andrews et al., 1989; Ey et al., 1993; Meloni et al., 1995). Several classification methods have divided G. duodenalis isolates into three major groups (Baruch et al., 1996), and our consistent classification of strains Portland-1 and WB in a same group confirmed previous results from other research groups (Baruch et al., 1996; Nash et al., 1985; Weiss et al., 1992), demonstrating the reliability of this quick RAPD technique in molecular characterization of G. duodenalis strains.
4. Conclusion The simplified genomic DNA extraction methods can readily be used to prepare DNA template for RAPD and probably other PCR-based DNA amplifi-
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Fig. 4. Evaluation of RAPD primers and comparison of RAPD patterns of G. duodenalis strains. AP-PCR parameters (in a total of 25 ml reaction mixture) were: 2.0 mM of MgCl 2 , 200 mM of each of four dNTPs, 2.5 U of Taq DNA polymerase, template DNA from 5 3 10 4 trophozoites by freezing-thawing treatment. Lane M, DNA size marker; lane 1 and 6, Portland-1 strain; lane 2 and 7, WB strain; lane 3 and 8, KS strain; lane 4 and 9, New Orleans-1 strain; lane 5 and 10, UNO / 04 / 87 / 1 strain. A. Primer GPD51 (lanes 1–5) and GPD61 (lanes 6–10); B. Primer GPD52 and GPD62; C. Primer GPD53 and GPD63; D. Primer GPD71 and GPD72. E. Primer GPD81 and GPD82.
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cation fingerprinting. They will be especially useful when many samples are to be analyzed, since little manipulation is involved. Under optimized AP-PCR conditions, reproducible and distinguishable fingerprints of human origin G. duodenalis stains were generated by five newly designed RAPD primers, demonstrating their effectiveness in DNA fingerprinting of G. duodenalis. The consistent classification of strains Portland-1 and WB in the same group not only verified previous classification but also further demonstrated the reliability of the reported RAPD procedure.
Acknowledgements This work was supported by the Livestock Disease Research Laboratory, School of Veterinary Medicine, University of California, Davis. We thank Tadesse W. Mariam for laboratory assistance.
References Abed, Y., Davin-Regli, A., Charrel, R.N., Bollet, C., Demicco, P., 1995. Variation of RAPD fingerprint patterns using different DNA-extraction methods with gram-positive bacteria. World J. Microbiol. Biotech. 11, 238–239. Andrews, R.H., Adams, M., Boreham, P.F.L., Mayrhofer, G., Meloni, B.P., 1989. Giardia intestinalis: electrophoretic evidence for a species complex. Int. J. Parasitol. 19, 183–190. Baruch, A.C., Isaac-Renton, J., Adam, R.D., 1996. The molecular epidemiology of Giardia lamblia: a sequence-based approach. J. Infect. Dis. 174, 233–236. Craun, G.F., 1990. Waterborne giardiasis. In: Meyer, E.A. (Ed.), Giardiasis, Elsevier, Amsterdam, pp. 267–290. Davin-Regli, A., Abed, Y., Charrel, R.N., Bollet, C., deMicco, P., 1995. Variations in DNA concentrations significantly affect the reproducibility of RAPD fingerprint patterns. Res. Microbiol. 146, 561–568. Deng, M.Q., Cliver, D.O., 1998. Differentiation of Cryptosporidium parvum isolates by a simplified randomly amplified polymorphic DNA technique. Appl. Environ. Microbiol. 64, 1954–1957. Deng, M.Q., Cliver, D.O., Mariam, T.W., 1997. Immunomagnetic capture PCR (IC-PCR) to detect viable Cryptosporidium parvum oocysts from environmental samples. Appl. Environ. Microbiol. 63, 3134–3138. Ey, P.L., Darby, J.M., Andrews, R.H., Mayrhofer, G., 1993. Giardia intestinalis: detection of major genotypes by restriction analysis of gene amplification products. Int. J. Parasitol. 23, 591–600.
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Hadrys, H., Balick, M., Schierwater, B., 1992. Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Mol. Ecol. 1, 55–63. Homan, W.L., vanEnckevort, F., Limper, L., vanEijs, G., Schoone, G., Kasperzak, W., Majewska, A., vanKnapen, F., 1992. Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes. Parasitol. Res. 78, 316–323. Homan, W.L., Gilsing, M., Bentala, H., Limper, L., vanKnapen, F., 1998. Characterization of Giardia duodenalis by polymerase-chain-reaction fingerprinting. Parasitol. Res. 84, 707– 714. Keister, D.B., 1983. Axenic culture of Giardia lamblia in TYI-S33 medium supplemented with bile. Trans. Roy. Soc. Trop. Med. Hyg. 77, 487–488. LeChevallier, M.W., Norton, W.D., 1995. Giardia and Cryptosporidium in raw and finished water. J. Am. Water. Works Assoc. 87, 54–68. McRoberts, K.M., Meloni, B.P., Morgan, U.M., Marano, R., Binz, N., Erlandsen, S.L., 1996. Morphological and molecular characterization of Giardia isolated from the straw-necked ibis (Threskiornis spinicollis) in western Australia. J. Parasitol. 82, 711–718. Meloni, B.P., Lymbery, A.J., Thompson, R.C.A., 1995. Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: implications for reproductive biology, population structure, taxonomy and epidemiology. J. Parasitol. 81, 368–383. Monis, P.T., Mayrhofer, G., Andrews, R.H., Homan, W.L., Limper, L., Ey, P.L., 1996. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology 112 (Pt 1), 1–12. Monis, P.T., Andrews, R.H., 1998. Molecular epidemiology: assumptions and limitations of commonly applied methods. Int. J. Parasitol. 28, 981–987. Morgan, U.M., Constantine, C., Greene, W.K., Thompson, R.C.A., 1993. RAPD (random amplified polymorphic DNA) analysis of Giardia DNA and correlation with isoenzyme analysis. Trans. Roy. Soc. Trop. Med. Hyg. 87, 702–705. Nash, T.E., 1992. Surface antigen variability and variation in Giardia lamblia. Parasitol. Today 8, 29–234. Nash, T.E., McCutchan, T., Keister, D., Dame, J.B., Conrad, J.D., Gillin, F.D., 1985. Restriction endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. J. Infect. Dis. 152, 64–73. Neto, E.D, Caballero, O.L., Vidigal, T.H., Pena, S.D., Simpson, A.J., 1997. Partially degraded DNA of parasitological interest serves as an adequate template for the production of random amplified polymorphic DNAs (RAPDs). J. Parasitol 83, 753– 775. Proctor, E.M., Issac-Renton, J.L., Boyd, J., Wong, Q., Bowie, W.R., 1989. Isoenzyme analysis of human and animal isolates of Giardia duodenalis from British Columbia. Canada. Am. J. Trop. Med. Hyg. 41, 411–415. Sarafis, K., Issac-Renton, J., 1993. Pulse-field gel electrophoresis as a method of biotyping of Giardia duodenalis. Am. J. Trop. Med. Hyg. 48, 134–144. VanBelkum, A., Homan, W., Limper, L., Quint, W.G.V., 1993.
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Genotyping isolates and clones of Giardia duodenalis by polymerase chain reaction: implications for the detection of genetic variation among protozoan parasite species. Mol. Biochem. Parasitol. 61, 69–77. Van Keulen, H., Homan, W.L., Erlandsen, S.L., Jarroll, E.L., 1995. A three nucleotide signature sequence in small subunit rRNA divides human Giardia in two different genotypes. J. Eukaryot. Microbiol. 42, 392–394.
Weiss, J.B., vanKeulen, H., Nash, T.E., 1992. Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequence using the polymerase chain reaction. Mol. Biochem. Parasitol. 54, 73–86. Williams, J.G.K., Hanafey, M.K., Rafalski, J.A., Tingey, S.V., 1993. Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol. 218, 704–740.
Journal of Microbiological Methods
Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis Ming-Qi Deng, Dean O. Cliver* Department of Population Health and Reproduction, School of Veterinary Medicine, University of California at Davis, One Shields Ave., Davis, CA 95616 -8743, USA Received 16 February 1999; received in revised form 15 April 1999; accepted 26 April 1999
Abstract A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains. 1999 Elsevier Science B.V. All rights reserved. Keywords: Giardia duodenalis; RAPD; AP-PCR; DNA extraction; Fingerprinting
1. Introduction The protozoan parasite Giardia duodenalis (synonyms: G. intestinalis or G. lamblia) is a commonly reported waterborne pathogen capable of causing prolonged gastrointestinal disease in infected humans and various mammals (LeChevallier and Norton, 1995). It occurs throughout the world in both developed countries and developing areas (Craun, 1990). However, the taxonomic criteria of G. duodenalis based on host specificity and cell *Corresponding author. Tel.: 1 1-530-754-9120; fax: 1 1-530752-5845. E-mail address: [email protected] (D.O. Cliver)
morphology have been controversial, as extensive variation in G. duodenalis has been reported from various studies. G. duodenalis strains / isolates of different origins have been characterized using methods such as isoenzyme electrophoretic analysis, antigenic comparison, restriction endonuclease analysis, and pulsed field gel electrophoresis analysis (Andrews et al., 1989; Homan et al., 1992; Nash, 1992; Nash et al., 1985; Proctor et al., 1989; Sarafis and Issac-Renton, 1993). In the past few years, polymerase-chain reaction (PCR)-based fingerprinting methods requiring smaller quantities of DNA and less parasite material have also been applied, including amplification and sequencing of a 183-bp region of the 18S rRNA gene (Weiss et al., 1992), sequence
0167-7012 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 99 )00067-6
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M. Deng, D.O. Cliver / Journal of Microbiological Methods 37 (1999) 193 – 200
analysis of the 59-end of the 16S rRNA following amplification (Van Keulen et al., 1995), restriction fragment length polymorphisms (RFLP) of a glutamate dehydrogenase gene segment or other genomic region (Homan et al., 1998; Monis et al., 1996; Monis and Andrews, 1998), and randomly amplified polymorphic DNA (RAPD) analysis (McRoberts et al., 1996; Meloni et al., 1995; Morgan et al., 1993; Van Belkum et al., 1993). RAPD differs from normal PCR in that a single arbitrary primer is used instead of two sequencespecific primers. Compared to other PCR-based fingerprinting methods, RAPD requires no sequence information of a particular genomic region and there is no cloning or sequencing (Hadrys et al., 1992; Williams et al., 1993). Although RAPD is susceptible to contaminant-organism or foreign DNA, this has not been a major concern for G. duodenalis, since trophozoites from axenic cultures of G. duodenalis strains were generally used for molecular characterization. By applying the RAPD technique, it has been shown that axenic isolates of G. duodenalis from different locations could be differentiated and classified into different groups, corresponding to the classification by other techniques (McRoberts et al., 1996; Meloni et al., 1995; Morgan et al., 1993; Van Belkum et al., 1993). Our goals in this study were to establish a rapid RAPD procedure by applying quick genomic DNA preparation methods and to evaluate new arbitrary primers to facilitate molecular characterization of G. duodenalis strains.
2. Materials and methods
2.1. G. duodenalis strains Trophozoites of five human-origin G. duodenalis strains, isolated from clinical samples of various sources and cultured axenically for long terms, were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA): Portland-1 (ATCC 30888), WB (30957), KS (50114), New Orleans-1 (50137), and UNO / 04 / 87 / 1 (50184). They were grown axenically in borosilicate tubes at 378C in Keister modified Diamond’s TYI-S-33 medium as previously described (Keister, 1983). After they had grown to form a monolayer, trophozoites were
detached from the borosilicate tube surfaces by chilling in ice-water for 15 min, pelleted by centrifugation at 500 3 g for 10 min, washed once with distilled water and once with 1 3 Tris-EDTA buffer (TE, containing 10 mM Tris base and 1 mM EDTA, pH 8.0), enumerated under a hemacytometer, and resuspended in 1 3 TE to the concentration of 1.0 3 10 7 / ml. The suspension containing 1 3 10 6 trophozoites (100 ml) was then used for genomic DNA preparation as described below.
2.2. Extraction of genomic DNA Two simple methods were applied. The freezingthawing treatment was done by immersing 0.5 ml centrifuge tubes containing 100 ml of trophozoite suspension in liquid nitrogen for 2 min and then incubating in a 658C waterbath for 2 min, which was repeated five times; the supernatant following a 5min centrifugation at 10 000 3 g was stored at 2 208C. Sonication was carried out by incubating 0.5 ml tubes containing the trophozoite suspension in a waterbath-type sonicator (Branson B-52 ultrasonic cleaner, Branson Cleaning Equipment Company, Shelton, CT, USA) filled with ice-water for 15 s (short time sonication) or 5 min (long time sonication); the supernatant following a 5-min centrifugation at 10 000 3 g was stored at 2 208C. To evaluate the efficiencies of these two methods, genomic DNA was also extracted from trophozoites using a conventional DNA isolation method involving proteinase K digestion, phenol / chloroform / isoamyl alcohol extraction, and isopropanol precipitation, as was described elsewhere (Deng et al., 1997). Extracted DNA was eventually resuspended in 100 ml 1 3 TE and stored at 2 208C until further analysis.
2.3. RAPD primers A total of 10 RAPD primers were designed: GPD51 (AGC GTA CAC T), GPD52 (ATT GCG TCG A), GPD53 (GCT TGT GAA C), GPD61 (GCT TGT GAA C), GPD62 (CAA TGC CCG A), GPD63 (CAA TGC CCG A), GPD71 (TGC GCA GCT G), GPD72 (ACG AGC GTG G), GPD81 (CCA GCG TCG C), and GPD82 (GGC CGA CCG T). They are all 10 nucleotides long, with 50–80%
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G–C content, and contain no palindromic sequence. GPD62 and GPD63 had been used in an earlier study, in which both primers demonstrated their abilities in differentiating isolates of another protozoan parasite, Cryptosporidium parvum (Deng and Cliver, 1998).
2.4. Optimization of arbitrarily primed-polymerase chain reaction (AP-PCR) The PCR reaction mixture (25 ml) contained 1 3 PCR Buffer II (Perkin-Elmer Cetus Corp., Norwalk, CT, USA), MgCl 2 solution (Perkin-Elmer), each of the four deoxynucleoside triphosphates (dNTPs, Perkin-Elmer), a 10-nucleotide primer (GeneMed Biotechnologies, San Francisco, CA, USA), 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer), and template DNA suspension. It was overlaid with 25 ml of mineral oil to prevent evaporation. PCR amplification was performed on a Progene 120 Thermal Cycler (Techne Inc., Princeton, NJ, USA), using a standard RAPD thermal cycling program: after denaturation at 948C for 3 min, the reaction mixtures underwent 45 cycles of denaturation at 948C for 1 min, annealing at 368C for 1 min, and extension at 728C for 2 min, with an additional 7-min extension at 728C. AP-PCR parameters were optimized using primer GPD62, strain Portland-1, and genomic DNA extracted by freezing-thawing treatment. The MgCl 2 concentration was altered from 2.0 to 8.0 mM, primer concentration varied from 0.25 to 2.0 mM, and dNTP concentrations ranged from 50 to 400 mM. The optimal amount of template DNA in a 25 ml reaction was determined by varying the volume of extracted genomic DNA suspension and referring it to the trophozoite numbers, which ranged from 5 3 10 3 to 1.5 3 10 6 . Following AP-PCR, 20 ml of product was separated on 1.8% agarose gel, stained with 0.5 mg / ml ethidium bromide, and examined under a UV-transilluminator. A 1.0 kb DNA ladder (GIBCO BRL Life Technologies, Gaithersburg, MD, USA) was included in electrophoresis as a size marker. To ensure the RAPD reproducibility, all AP-PCR reactions were repeated three times; a successful RAPD was defined as one with multiple distinctive
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bands and with identical profiles from reactions of different batches.
2.5. Evaluation of primers and comparison of RAPD patterns of G. duodenalis strains Conditions chosen as optimum were used in all subsequent analyses for primer evaluation and comparison of RAPD patterns of the five G. duodenalis strains. Primers were evaluated as useful in the assay by two criteria: (1) they exhibited polymorphisms among different strains; and (2) the polymorphisms were reproducible among samples of the same strain. RAPD patterns were compared for the number, size, and intensity of major (distinctive) bands. Differences in intensities of minor bands were neglected.
3. Results and discussion
3.1. Optimization of AP-PCR parameters With the MgCl 2 concentrations escalating from 2.0 mM to 4.0 mM (Fig. 1, lanes 1–5), more
Fig. 1. RAPD optimization: MgCl 2 concentration. Other AP-PCR parameters (in a total of 25 ml reaction mixture) were: 200 mM of each of four dNTPs, 1.0 mM of GPD62, 2.5 U of Taq DNA polymerase, and template DNA from 5 3 10 4 trophozoites of Portland-1 strain by freezing-thawing treatment. Lane M, DNA size marker (1 kb ladder), the arrow-headed bands (from top to bottom) are approximately 2.0, 1.6, 1.0, and 0.5 kb, respectively; lanes 1–9, patterns obtained at MgCl 2 concentration 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0 mM, respectively.
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amplification products were obtained; and the intensities of the small-size bands ( , 0.8 kb) increased, while the intensities of large-size bands decreased. MgCl 2 concentrations over 4.0 mM had little further effect on the number of products; however, the intensities of larger size bands decreased (Fig. 1, lanes 6–9) and the RAPD profile became less reproducible, probably because amplification became less specific under high concentrations of Mg 11 . Therefore, the optimal concentration of MgCl 2 was determined as 4.0 mM. Similarly, the optimal concentrations of dNTP and primer were determined as 200 mM and 1.0 mM respectively. The optimal template amount for a single tube (25 ml) reaction was determined as the amount of DNA prepared from 5 3 10 4 trophozoites. As shown in Fig. 2, more distinctive bands were obtained and the intensities of the bands were higher from such a trophozoite number (lane 4). When more template was used, fewer bands with higher intensities were generated (lanes 1–3). On the other hand, using less template usually resulted in more small size bands (especially bands , 0.5 kb) with reduced intensities (lanes 5–6), and the product profile was less reproducible (data not shown). This agrees with a previous observation of RAPD reproducibility affect-
Fig. 2. RAPD optimization: template amount. Other AP-PCR parameters (in a total of 25 ml reaction mixture) were: 2.0 mM of MgCl 2 , 200 mM of each of four dNTPs, 1.0 mM of GPD62, 2.5 U of Taq DNA polymerase. Lane M, 1 kb DNA ladder; lanes 1–6, template DNA from Portland-1 strain prepared by freezing-thawing treatment from 1.5 3 10 6 , 5 3 10 5 , 1 3 10 5 , 5 3 10 4 , 1.5 3 10 4 , 5 3 10 3 trophozoites respectively.
ed by template DNA concentrations (Davin-Regli et al., 1995).
3.2. Comparison of DNA extraction methods As shown in Fig. 3, similar RAPD profiles were obtained by all three methods and they were all reproducible, indicating that methods of DNA preparation had no effect on RAPD. Although there were minor differences in RAPD profiles when DNA extracted by sonication was used, the differentiation ability of RAPD is unlikely to be affected, because only absence or presence of distinctive bands and major differences in intensities of those bands matter in comparing RAPD profiles. Although the proteinase K–phenol method usually generated bands with somewhat higher intensities (lanes 3–4), the main disadvantage of this method is the intensive hands-on manipulations and the time required for the process, which becomes tedious when a large number of samples are to be analyzed. In contrast both the freezing–thawing treatment (lanes 1–2) and sonication (either short-time or long-time, lanes 5–6 and lanes 7–8) can be completed within 30 min and there is little manipulation involved, minimizing possible
Fig. 3. Comparison of genomic DNA preparation methods. APPCR parameters (in a total of 25 ml reaction mixture) were: 2.0 mM of MgCl 2 , 200 mM of each of four dNTPs, 1.0 mM of GPD62, 2.5 U of Taq DNA polymerase, and genomic DNAs from 5 3 10 4 trophozoites of Portland-1 strain. Lane M, DNA size marker; lanes 1–2, DNA prepared by freezing-thawing treatment; lanes 3–4, DNA prepared by proteinase K–phenol method; lanes 5–6, DNA prepared by short-time (15 s) sonication; lanes 7–8, DNA prepared by long-time (5 min) sonication.
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accidental contamination of samples during the genomic DNA extraction process. Although sonication can partially degrade DNA, the insensitivity of RAPD to different DNA extraction methods observed in this study demonstrated that degraded DNA can be used in RAPD, echoing a previous report that reproducible and complex band patterns could be obtained from RAPD using partially degraded parasite (Schistosoma mansoni) DNA (Neto et al., 1997). However, our results differred from another report that different DNA extraction methods (Chelex, microwave and phenol / chloroform method) led to significantly different RAPD patterns of gram-positive bacteria Staphylococcus epidermidis and Streptococcus faecalis (Abed et al., 1995). In a subsequent investigation, genomic DNAs extracted by freezing–thawing treatment or longtime (5 min) sonication were applied to normal PCR using several sequence-specific primer pairs. Consistently, a product of the expected length - ranging from 171 bp to 1852 bp and correspondent to the specific primer pair, was obtained (data not shown here); suggesting that these simple DNA extraction methods are also effective in preparing templates for other PCR-based fingerprinting methods.
3.3. Evaluation of primers The results are shown in Fig. 4, panels A–E. Among the 10 primers investigated, GPD51 (Fig. 4A, lanes 1–5), GPD63 (Fig. 4C, lanes 6–10), and GPD71 (Fig. 4D, lanes 1–5) failed to produce polymorphisms among the G. duodenalis strains. GPD53 (Fig. 4C, lanes 1–5) and GPD62 (Fig. 4B, lanes 6–10) produced polymorphisms, but the polymorphisms were not reproducible. The remaining five primers, GPD52 (Fig. 4B, lanes 1–5), GPD61 (Fig. 4A, lanes 6–10), GPD72 (Fig. 4D, lanes 6–10), GPD81, and GPD82 (Fig. 4E) all produced reproducible polymorphisms and thus have the potential to be used in RAPD analysis for strain differentiation.
3.4. Comparison of RAPD patterns of G. duodenalis strains This was based on comparison of RAPD profiles generated from primers GPD 52, 61, 72, 81, and 82.
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Regardless of the primer used, profiles of strains Portland-1 and WB were always identical, indicating that the belong to the same group. The profiles of strains KS and UNO / 04 / 87 / 1 were also identical, with the only exception being the difference in intensities of a | 1.4 kb band generated by GDP61 (weak in strain KS, strong in UNO / 04 / 87 / 1) (Fig. 4A, lanes 8 and 10), suggesting that they are in the same group. They were readily distinguishable from profiles of strains of Portland-1 and WB, indicating that they belong to a different group. Profiles of strain New Orleans-1 and those of KS and UNO / 04 / 87 / 1 were always distinguishable, regardless the primer used in AP-PCR, indicating that New Orleans-1 should be in a different group from strains KS and UNO / 04 / 87 / 1. In contrast, the profile of New Orleans-1 and those or Portland-1 / WB had high similarities when primers GPD 72, 81, and 82 were used and were distinguishable when GPD 52 and 61 were used. Therefore, these five G. duodenalis strains seemed to belong to at least two groups: one consists of strains KS and UNO / 04 / 87 / 1, while the other consists of strains Portland-1 and WB. The New Orleans-1 strains could be in the same group as Portland-1 and WB or a third group. Although a very limited number of isolates were used in this study, apparent genetic diversity was observed even among these G. duodenalis strains, which were all from human sources. This showed the possible existence of morphologically similar but genetically distinct (cryptic) species within G. duodenalis (Andrews et al., 1989; Ey et al., 1993; Meloni et al., 1995). Several classification methods have divided G. duodenalis isolates into three major groups (Baruch et al., 1996), and our consistent classification of strains Portland-1 and WB in a same group confirmed previous results from other research groups (Baruch et al., 1996; Nash et al., 1985; Weiss et al., 1992), demonstrating the reliability of this quick RAPD technique in molecular characterization of G. duodenalis strains.
4. Conclusion The simplified genomic DNA extraction methods can readily be used to prepare DNA template for RAPD and probably other PCR-based DNA amplifi-
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Fig. 4. Evaluation of RAPD primers and comparison of RAPD patterns of G. duodenalis strains. AP-PCR parameters (in a total of 25 ml reaction mixture) were: 2.0 mM of MgCl 2 , 200 mM of each of four dNTPs, 2.5 U of Taq DNA polymerase, template DNA from 5 3 10 4 trophozoites by freezing-thawing treatment. Lane M, DNA size marker; lane 1 and 6, Portland-1 strain; lane 2 and 7, WB strain; lane 3 and 8, KS strain; lane 4 and 9, New Orleans-1 strain; lane 5 and 10, UNO / 04 / 87 / 1 strain. A. Primer GPD51 (lanes 1–5) and GPD61 (lanes 6–10); B. Primer GPD52 and GPD62; C. Primer GPD53 and GPD63; D. Primer GPD71 and GPD72. E. Primer GPD81 and GPD82.
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cation fingerprinting. They will be especially useful when many samples are to be analyzed, since little manipulation is involved. Under optimized AP-PCR conditions, reproducible and distinguishable fingerprints of human origin G. duodenalis stains were generated by five newly designed RAPD primers, demonstrating their effectiveness in DNA fingerprinting of G. duodenalis. The consistent classification of strains Portland-1 and WB in the same group not only verified previous classification but also further demonstrated the reliability of the reported RAPD procedure.
Acknowledgements This work was supported by the Livestock Disease Research Laboratory, School of Veterinary Medicine, University of California, Davis. We thank Tadesse W. Mariam for laboratory assistance.
References Abed, Y., Davin-Regli, A., Charrel, R.N., Bollet, C., Demicco, P., 1995. Variation of RAPD fingerprint patterns using different DNA-extraction methods with gram-positive bacteria. World J. Microbiol. Biotech. 11, 238–239. Andrews, R.H., Adams, M., Boreham, P.F.L., Mayrhofer, G., Meloni, B.P., 1989. Giardia intestinalis: electrophoretic evidence for a species complex. Int. J. Parasitol. 19, 183–190. Baruch, A.C., Isaac-Renton, J., Adam, R.D., 1996. The molecular epidemiology of Giardia lamblia: a sequence-based approach. J. Infect. Dis. 174, 233–236. Craun, G.F., 1990. Waterborne giardiasis. In: Meyer, E.A. (Ed.), Giardiasis, Elsevier, Amsterdam, pp. 267–290. Davin-Regli, A., Abed, Y., Charrel, R.N., Bollet, C., deMicco, P., 1995. Variations in DNA concentrations significantly affect the reproducibility of RAPD fingerprint patterns. Res. Microbiol. 146, 561–568. Deng, M.Q., Cliver, D.O., 1998. Differentiation of Cryptosporidium parvum isolates by a simplified randomly amplified polymorphic DNA technique. Appl. Environ. Microbiol. 64, 1954–1957. Deng, M.Q., Cliver, D.O., Mariam, T.W., 1997. Immunomagnetic capture PCR (IC-PCR) to detect viable Cryptosporidium parvum oocysts from environmental samples. Appl. Environ. Microbiol. 63, 3134–3138. Ey, P.L., Darby, J.M., Andrews, R.H., Mayrhofer, G., 1993. Giardia intestinalis: detection of major genotypes by restriction analysis of gene amplification products. Int. J. Parasitol. 23, 591–600.
199
Hadrys, H., Balick, M., Schierwater, B., 1992. Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Mol. Ecol. 1, 55–63. Homan, W.L., vanEnckevort, F., Limper, L., vanEijs, G., Schoone, G., Kasperzak, W., Majewska, A., vanKnapen, F., 1992. Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes. Parasitol. Res. 78, 316–323. Homan, W.L., Gilsing, M., Bentala, H., Limper, L., vanKnapen, F., 1998. Characterization of Giardia duodenalis by polymerase-chain-reaction fingerprinting. Parasitol. Res. 84, 707– 714. Keister, D.B., 1983. Axenic culture of Giardia lamblia in TYI-S33 medium supplemented with bile. Trans. Roy. Soc. Trop. Med. Hyg. 77, 487–488. LeChevallier, M.W., Norton, W.D., 1995. Giardia and Cryptosporidium in raw and finished water. J. Am. Water. Works Assoc. 87, 54–68. McRoberts, K.M., Meloni, B.P., Morgan, U.M., Marano, R., Binz, N., Erlandsen, S.L., 1996. Morphological and molecular characterization of Giardia isolated from the straw-necked ibis (Threskiornis spinicollis) in western Australia. J. Parasitol. 82, 711–718. Meloni, B.P., Lymbery, A.J., Thompson, R.C.A., 1995. Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: implications for reproductive biology, population structure, taxonomy and epidemiology. J. Parasitol. 81, 368–383. Monis, P.T., Mayrhofer, G., Andrews, R.H., Homan, W.L., Limper, L., Ey, P.L., 1996. Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology 112 (Pt 1), 1–12. Monis, P.T., Andrews, R.H., 1998. Molecular epidemiology: assumptions and limitations of commonly applied methods. Int. J. Parasitol. 28, 981–987. Morgan, U.M., Constantine, C., Greene, W.K., Thompson, R.C.A., 1993. RAPD (random amplified polymorphic DNA) analysis of Giardia DNA and correlation with isoenzyme analysis. Trans. Roy. Soc. Trop. Med. Hyg. 87, 702–705. Nash, T.E., 1992. Surface antigen variability and variation in Giardia lamblia. Parasitol. Today 8, 29–234. Nash, T.E., McCutchan, T., Keister, D., Dame, J.B., Conrad, J.D., Gillin, F.D., 1985. Restriction endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. J. Infect. Dis. 152, 64–73. Neto, E.D, Caballero, O.L., Vidigal, T.H., Pena, S.D., Simpson, A.J., 1997. Partially degraded DNA of parasitological interest serves as an adequate template for the production of random amplified polymorphic DNAs (RAPDs). J. Parasitol 83, 753– 775. Proctor, E.M., Issac-Renton, J.L., Boyd, J., Wong, Q., Bowie, W.R., 1989. Isoenzyme analysis of human and animal isolates of Giardia duodenalis from British Columbia. Canada. Am. J. Trop. Med. Hyg. 41, 411–415. Sarafis, K., Issac-Renton, J., 1993. Pulse-field gel electrophoresis as a method of biotyping of Giardia duodenalis. Am. J. Trop. Med. Hyg. 48, 134–144. VanBelkum, A., Homan, W., Limper, L., Quint, W.G.V., 1993.
200
M. Deng, D.O. Cliver / Journal of Microbiological Methods 37 (1999) 193 – 200
Genotyping isolates and clones of Giardia duodenalis by polymerase chain reaction: implications for the detection of genetic variation among protozoan parasite species. Mol. Biochem. Parasitol. 61, 69–77. Van Keulen, H., Homan, W.L., Erlandsen, S.L., Jarroll, E.L., 1995. A three nucleotide signature sequence in small subunit rRNA divides human Giardia in two different genotypes. J. Eukaryot. Microbiol. 42, 392–394.
Weiss, J.B., vanKeulen, H., Nash, T.E., 1992. Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequence using the polymerase chain reaction. Mol. Biochem. Parasitol. 54, 73–86. Williams, J.G.K., Hanafey, M.K., Rafalski, J.A., Tingey, S.V., 1993. Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol. 218, 704–740.