JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/00/$04.0010

Feb. 2000, p. 737–744 Vol. 38, No. 2

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Differential Diagnosis of Taenia saginata and Taenia soliumInfection by PCR

LUIS MIGUEL GONZALEZ,1 ESTRELLA MONTERO,1 LESLIE J. S. HARRISON,2*R. MICHAEL E. PARKHOUSE,3 AND TERESA GARATE1

Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Centro Nacional de Microbiologia, 28220Majadahonda, Madrid, Spain1; University of Edinburgh, Centre for Tropical Veterinary Medicine, Easter Bush, Roslin,Midlothian, Scotland EH25 9RG2; and Institute for Animal Health, Pirbright Laboratories, Pirbright, Woking, Surrey,

England GU24 0NF3

Received 16 June 1999/Returned for modification 21 July 1999/Accepted 3 November 1999

We have designed species-specific oligonucleotides which permit the differential detection of two species ofcestodes, Taenia saginata and Taenia solium. The oligonucleotides contain sequences established for twopreviously reported, noncoding DNA fragments cloned from a genomic library of T. saginata. The first, whichis T. saginata specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 timesin direct tandem along the 1,272-bp fragment. From this sequence the two oligonucleotides that were selected(oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from T. saginata but notT. solium or other related cestodes and had a sensitivity down to 10 pg of T. saginata gDNA. The second DNAfragment (fragment HDP2; 3,954 bp) hybridized to both T. saginata and T. solium DNAs and was not arepetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) de-signed from the sequence of HDP2 allowed the differential amplification of gDNAs from T. saginata, T. solium,and Echinococcus granulosus in a multiplex PCR, which exhibits a sensitivity of 10 pg.

Taenia saginata and Taenia solium are the two taeniids ofgreatest economic and medical importance, causing bovineand porcine cysticercosis and taeniasis in humans. In addition,T. solium eggs can infect humans, often giving rise to fatalneurocysticercosis (12, 39). Infections with these cestodes aretherefore a serious public health problem in areas of endemic-ity. In addition, an increase in the number of cases in areas ofnonendemicity has been observed in recent years (36).

At present, there is no rapid, facile means of diagnosis ofhuman taeniasis and there is an obvious need for sensitive andspecific differential tests for T. solium and T. saginata detectionand interruption of human cysticercosis transmission. Conven-tional coproscopical examination has a low specificity and sen-sitivity (29), whereas coproantigen detection by enzyme-linkedimmunosorbent assay, although sensitive, suffers from poorspecificity due to cross-reactions with other taeniids and re-lated helminths (1, 8, 23, 24). A recently developed Westernblot assay measures antibody to adult Taenia and thus does notnecessarily detect an active infection (43). Furthermore, thisassay requires the preparation of secreted antigens from im-mature adult tapeworms recovered from immunosuppressedhamsters, which is impractical for routine use. The use of DNAprobes, as successfully used for species-specific detection ofvarious parasites (2, 11, 14, 35, 37, 44), including T. solium andT. saginata (5, 13, 19, 32), is time-consuming and relativelyinsensitive. More recently, however, PCR with oligonucleotideprimers derived from such species-specific probes (15, 16, 26,27) has provided a truly rapid and sensitive method for theidentification of helminth parasites in general.

This paper describes the design of oligonucleotides, basedon the sequences of two previously described diagnostic DNAtests (19), which permitted positive identification of T. saginata

and T. solium. The first DNA probe, probe HDP1, is a repet-itive sequence that yielded PCR probes specific for T. saginata,while the second sequence, probe HDP2, yielded a multiplexPCR probes which allowed the simultaneous identification ofT. solium, T. saginata, and Echinococcus granulosus.

MATERIALS AND METHODS

Extraction and sources of DNA. Genomic DNAs (gDNAs) of T. saginata, T.solium, Taenia taeniformis (Belgian isolate), T. taeniformis (Malaysian isolate),and E. granulosus were obtained by a phenol extraction and ethanol precipitationprotocol (34). Bovine and human DNAs were purchased commercially (SigmaChemical Company, St. Louis, Mo.).

Subcloning strategy. The HDP1 and HDP2 genomic sequences were clonedfollowing the differential screening of a T. saginata lgt10 genomic library (19).Since one of the HDP1 EcoRI restriction enzyme digestion sites was damaged,the HDP1 fragment was isolated from the recombinant phage by EcoRI-BamHI(Promega Corporation, Madison, Wis.) digestion. A 5,100-bp fragment whichwas composed of a 1,272-bp fragment of T. saginata gDNA and a 3,800-bp frag-ment from the short arm of lgt10 phage was obtained. The insert was subclonedinto the EcoRI and BamHI restriction sites of pBluescript KS1 (Stratagene, LaJolla, Calif.), and the recombinant plasmid (pBluescript KS1, lgt10 fragment,HDP1) was named pPTs4. The HDP2 sequence was isolated from the recombi-nant phage by EcoRI digestion (Promega Corporation), yielding a 3,954-bp frag-ment which was subcloned into the EcoRI site of pBluescript KS1 (Stratagene,La Jolla, Calif.).

HDP1 and HDP2 sequencing. A designated progressive unidirectional erasestrategy (Promega Corporation) was used in order to sequence the T. saginataDNA inserts. Sequencing of HDP1 and HDP2 was carried out with two auto-mated sequencing systems: fluorescence-based labeling with the ABI PRISMsystem (Perkin-Elmer, Langen, Germany) and the ALF system (Pharmacia,Uppsala, Sweden). The HDP1 and HDP2 DNA sequences were compared withthose available in the EMBL databank by using software packages from theGenetics Computer Group (9).

Slot blot hybridization. Samples of either gDNA or plasmid DNA were pre-pared after first diluting the DNA to the required concentrations and thendenaturation with 0.3 M NaOH and incubation at 80°C for 10 min, followed byneutralization with 0.25 M Tris-HCl (pH 7.5)–0.25 M HCl–12.53 SSC (13 SSCis 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]) buffer. The samples werethen transferred by vacuum onto nitrocellulose membranes with a slot blotmanifold apparatus (Shleicher & Schuell, Dassel, Germany), in accordance withthe manufacturer’s instructions.

Electrophoresis, Southern blotting, labeling, and hybridization procedures.The genomic organizations of the HDP1 and the HDP2 DNA sequences wereexamined as follows. First, 3-mg aliquots of T. saginata gDNA were digested to

* Corresponding author. Mailing address: University of Edinburgh,Centre for Tropical Veterinary Medicine, Easter Bush, Roslin, Mid-lothian, Scotland, EH25 9RG. Phone: 44-131-6506217. Fax: 44-131-6506217. E-mail: Leslie.Harrison@ed.ac.uk.

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completion with different restriction endonucleases (Amersham Life Science,Buckinghamshire, England; Boehringer Mannheim GmbH, Mannheim, Ger-many; Promega Corporation) by following the procedures recommended by themanufacturers. Electrophoresis of the digested DNA samples and subsequenttransfer to positively charged nylon membranes (Boehringer Mannheim GmbH)were carried out by standard procedures (40). The HDP1 and HDP2 DNAprobes were nonradioactively labeled with digoxigenin-11-dUTP (BoehringerMannheim GmbH) by a random oligonucleotide primer method, in accordancewith the manufacturer’s instructions. Hybridizations were conducted overnightunder high-stringency conditions at 68°C. After hybridization, the filters werewashed at 68°C for 10 min in 23 SSC–0.1% sodium dodecyl sulfate (SDS) andthen for a further 40 min in 0.13 SSC–0.1% SDS. The immunodetection wascarried out with antidigoxigenin conjugated with alkaline phosphatase, and theimmune complexes were visualized with the chemiluminescence substrate CSPD(Boehringer Mannheim GmbH) on X-ray film with an intensifying screen atroom temperature for 15 min, as described in the manufacturer’s instructions.

In order to identify unique sequences of HDP2 that do not occur in the T.solium genome, 5-mg samples of T. saginata and T. solium gDNAs were digestedto completion with the ClaI restriction endonuclease (Amersham Life Science),as recommended by the manufacturer. Southern blotting, probe labeling, andhybridization were carried out as described above. The probes used were threenonoverlapping fragments derived from the HDP2 sequence and were desig-nated 5PHDP2, IPHDP2, and 3PHDP2.

Design of HDP1 and HDP2 primers. DNA sequence analysis was carried outwith the Primer Select Lasergene program (DNASTAR Inc., Madison, Wis.).The HDP1 sequence was used to design two oligonucleotide primers, primersPTs4F1 (59-GCAGTGTGCTGAAGATGAATA-39) and PTs4R1 (59-GAATTTGGCTCTCACTGAATG-39). An internal primer, primer PTs4I1 (59-ATACTACCAAATCGCAT-39), was also prepared. The HDP2 sequence was used todesign three oligonucleotide primers, primers PTs7S5F1 (59-CAGTGGCATAGCAGAGGAGGAA-39), PTs7S35F2 (59-CTTCTCAATTCTAGTCGCTGTGGT-39), and PTs7S35R1 (59-GGACGAAGAATGGAGTTGAAGGT-39). Theprimers were synthesized by Gibco BRL.

DNA amplification. PCR with HDP1-based primers was performed in a totalvolume of 25 ml containing PCR buffer (PCR buffer I; Perkin-Elmer), 0.4%glycerol, each deoxynucleoside triphosphate (Pharmacia, Uppsala, Sweden) at aconcentration of 200 mM, 0.25 mM PTs4F1, and 2.5 U of Taq polymerase(Perkin-Elmer). PCR conditions were 94°C for 5 min (initial denaturation),followed by 35 cycles at 94°C for 1 min, 60°C for 30 s, 72°C for 30 s, and 72°C for10 min (final extension). PTs4R1 (0.5 mM) was added to the reaction mixture atthe 25th cycle. Multiplex PCR with HDP2-based primers was performed in atotal volume of 25 ml with PCR buffer (PCR buffer I; Perkin-Elmer) and finalconcentrations of 0.4% glycerol, each deoxynucleoside triphosphate (Pharmacia)at a concentration of 200 mM, 0.5 mM primer PTs7S35F1, 0.5 mM primerPTs7S35F2, 1 mM primer PTs7S35R1, and 2.5 U of Taq polymerase (Perkin-Elmer). Conditions for the multiplex PCR with HDP2-based primers were 94°Cfor 5 min (initial denaturation), followed by 35 cycles at 94°C for 1 min, 56.5°Cfor 30 s, 72°C for 30 s, and 72°C for 10 min (final extension). Amplifications werecarried out in a GeneAmp TM PCR System 2400 Thermocycler (Perkin-Elmer).The amplification products were separated on 2% agarose gels and were visu-alized under UV light by ethidium bromide staining.

Nucleotide sequence accession numbers. The HDP1 sequence was assignedaccession no. AJ133764 and the HDP2 sequence was assigned accession no.AJ133740 (EBI, EMBL GenBank, and DDJB database).

RESULTS

HDP1 and HDP2 sequencing. For sequencing, the HDP2DNA fragment was directly subcloned from lgt10 phage intothe pBluescript SK1 plasmid, and then 25 nested deletedclones were selected and sequenced. A different strategy wasnecessary for HDP1, as one of the two EcoRI digestion siteshad been lost from the recombinant phage (see Materials andMethods), and so five nested deleted clones were selected andsequenced to determine the full sequence of HDP2. The fullsequences of HDP1 and HDP2 are shown in Fig. 1 and Fig. 2,respectively.

HDP1 and HDP2 sequence analysis. The 1,272-bp nucleo-tide sequence of HDP1 was highly repetitive, with 24 se-quences, each of 53 bp, in tandem array. An unambiguousconsensus sequence was derived from the 24 monomeric mo-tifs (Fig. 1). All of the motifs were remarkably similar, with amaximum difference of only four bases. Strikingly, the sameadenine-to-guanine transition occurred at nucleotide 19 inthree of the motifs located in the 4th, 7th, and 17th positionswithin the sequence. The fourth mutation, which appeared inthe 18th motif, was a cytidine-to-guanine transversion. These

mutations yielded three new ScaI recognition sites and oneBglI recognition site within the mutated motifs. Thus, therewas only a 0.3% sequence divergence from the establishedconsensus sequence.

The HDP1 fragment had an A1T content of 55%. It showedinternal repeats as one direct repeat (1/19) of 6 bp and one of5 bp (2/29), two of 4 bp (3/39, 4/49), and three of invertedrepeats, one of 5 bp (5/59) and one of 4 bp (6/69 and 7/79). The3,954-bp HDP2 nucleotide sequence was nonrepetitive, withan A1T content of 45% and no significant internal repeats.Stop codons occurred frequently in both sequences, and there-fore, no open reading frames of significant length were iden-tified. Finally, no significant homologies were found with se-quences reported in either the GenBank or the EMBL databank.

Genomic organization of HDP1 and HDP2 probes in T.saginata genome. In order to study the genomic organization ofthe HDP1 and the HDP2 sequences, taeniid gDNA was di-gested to completion with several restriction enzymes, trans-

FIG. 1. Repetitive T. saginata HDP1 sequence (1,272 bp). Each monomericunit is represented by a line. Twenty-four repeats are included. Substitution pointmutations are indicated by an arrow below each mutation. The restriction en-zyme recognition sites are indicated by the lines. Direct and inverted internalrepeats are indicated by arrows above and below the consensus sequence (con),respectively.

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ferred to membranes, and hybridized with both HDP1 andHDP2 under high-stringency conditions. With the HDP1probe, different patterns were obtained depending on the re-striction enzyme used (Fig. 3). Thus, ScaI, EcoRI, and RsaI

digestions yielded a regular ladder pattern with hybridizationfragments of different sizes. In contrast, digestion of gDNAwith restriction enzymes specific for sequences not locatedwithin the HDP1 sequence (PstI, HindIII, SalI, XhoI, and

FIG. 2. Nonrepetitive T. saginata HDP2 sequence (3,954 bp). The restriction enzyme recognition sites are indicated by the lines.

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BamHI) yielded a single band that was larger than 23 kb andthat hybridized with the HDP1 probe. Taking into account thehybridization patterns, the HDP1 restriction map, and the se-quence information, we calculated that the 53-bp monomersare arranged in direct tandem arrays along 23 kb or more inthe T. saginata genome.

When similar experiments were done by Southern blottingwith the HDP2 probe, digestion of T. saginata gDNA with ClaI,PstI, EcoRI, and RsaI enzymes yielded different restrictionpatterns, depending on the enzyme used, and an irregularladder distribution. These data suggested that the HDP2 frag-ment did not contain repeated sequences within the T. saginatagenome (Fig. 4). It is important to note that complete digestionof T. saginata gDNA with all the enzymes mentioned abovewas confirmed by analyzing the digested samples by agarose gelelectrophoresis (data not shown).

Copy number of the 53-bp monomers in T. saginata genome.The copy number of the 53-bp monomer in the T. saginatagenome was determined by slot blot analysis (Fig. 5) by titrat-ing DNA purified from T. saginata metacestodes and from theHDP1-containing pBluescript KS1 plasmid pPTs4 (the HDP1sequence accounts for 15.9% of the recombinant plasmid) andusing the digoxigenin-labeled HDP1 sequence as the probe.

The slots containing 100 ng of T. saginata gDNA and 2.85 ngof pPTs4 DNA showed identical hybridization intensities,whereas the pBluescript KS1 nonrecombinant vector did nothybridize (data not shown). These results indicated that theHDP1 sequence represented approximately 0.4% of the T.saginata DNA. Assuming that the size of the T. saginata ge-nome is similar to that of the closely related organism E.granulosus (genome size, 1.5 3 108-bp) (32), we calculated11,321 repeats of the 53-bp monomer per haploid genome ofthe parasite.

Design of PCR primers derived from HDP1 and HDP2 se-quences. The two oligonucleotide primers (primers PTs4F1and PTs4R1) designed from the HDP1 sequence (Fig. 6A)were manually selected because the repetitive nature of theDNA sequence precluded use of the Primer Select Lasergeneprogram. The three oligonucleotide primers prepared fromthe HDP2 sequence (primers PTs7S35F1, PTs7S35F2, andPTs7S35R1) were designed after demonstrating that digestionof HDP2 with SphI and ClaI restriction endonucleases yieldedthree nonoverlapping fragments (fragments 5PHDP2, IPHDP2,and 3PHDP2) (Fig. 6B). When these were tested by Southernblotting with T. saginata and T. solium gDNAs digested withClaI, hybridization of T. solium DNA occurred with the frag-ment IPHDP2 and 3PHDP2 sequences but not with the frag-ment 5PHDP2 sequence (Fig. 7). As this suggested that the5PHDP2 sequence was not included in the T. solium ge-nome, we used the Primer Select Lasergene program to syn-thesize three primers (primers PTs7S35F1, PTs7S35F2, andPTs7S35R1). Primer PTs7S35F1 was based on the 5PHDP2sequence, and primers PTs7S35F2 and PTs7S35R1 were de-signed from the IPHDP2 sequence (Fig. 6B).

Design of a T. saginata species-specific PCR with HDP1-based primers. Use of conventional PCR protocols with theprimers described above yielded nonspecific results, probablydue to the repetitive nature of the sequences and the highdegree of complementarity between the forward and reverseprimers (see HDP1 and HDP2 sequence analysis). After test-ing a number of different protocols, we empirically observedthat the addition of primer PTs4R1 at 24 cycles after the initial

FIG. 3. Southern blot of T. saginata gDNA (3 mg) cleaved with variousrestriction enzymes (ScaI [lane 1], PstI [lane 2], HindIII [lane 3], EcoRI [lane 4],SalI [lane 5], XhoI [lane 6], BamHI [lane 7], and RsaI [lane 8]) and probed withthe digoxigenin-labeled T. saginata HDP1 probe.

FIG. 4. Southern blot of T. saginata gDNA (3 mg) cleaved with variousrestriction enzymes (ClaI [lane 1], PstI [lane 2], EcoRI [lane 3], and RsaI [lane 4])and probed with the digoxigenin-labeled HDP2 probe.

FIG. 5. Sensitivity of the T. saginata HDP1 probe. Dilutions of T. saginatagDNA (200 ng [slot 1a], 100 ng [slot 1b], 50 ng [slot 1c], 25 ng [slot 1d], 12.5 ng[slot 1e], 6.25 ng [slot 1f], 3 ng [slot 1g], 1.5 ng [slot 1h]) and pPTs4 recombinantplasmid DNA (50 ng [slot 2a], 10 ng [slot 2b], 5 ng [slot 2c], 2.5 ng [slot 2d], 1.25ng [slot 2e], 0.6 ng [slot 2f], 0.3 ng [slot 2g], 0.15 ng [slot 2h]) were probed in aslot blot system with the digoxigenin-labeled T. saginata HDP1 probe.

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addition of primer PTs4F1 greatly improved the specificity ofthe PCR, yielding a characteristic ladder pattern of 10 to 11bands, with an approximately 50-bp size difference betweenthem. The specificity of the amplifications was confirmed bySouthern blot hybridization with the internal primer PTs4I1

(59-ATACTACCAAATCGCAT-39) (data not shown). Wesuggest that the highly repetitive nature of the HDP1 sequenceis responsible for the observed sensitivity, despite the expectedreduction in amplification due to the late addition of one of theprimers.

FIG. 6. (A) Locations of the PTs4F1 and PTs4R1 oligonucleotide primers within the 1,272-bp T. saginata HDP1 repetitive DNA sequence. The 24 repetitive unitsare indicated by continuous arrows. The restriction enzyme sites are indicated by lines, and the PTs4F1 and PTs4R1 oligonucleotide primers are indicated by arrows.(B) Locations of probes 5PHDP2, 1PHDP2, and 3PHDP2 within the 3,954-bp sequence of the T. saginata and T. solium HDP2 genomic clone. The restriction enzymesites are indicated by the lines, and the probes (5PHDP2, 1PHDP2, and 3PHDP2) used in Southern blots assays are indicated by wider lines below the HDP2 DNAsequence. The locations of the PTs7S35F1, PTs7S35F2, and PTs7S35R1 oligonucleotide primers are indicated by arrows.

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The potential diagnostic properties of this modified PCRprotocol were evaluated with purified gDNAs from T. saginata,T. solium, and other related cestodes. With amounts of T.saginata and T. solium DNA in the 10- to 40-ng range, theladder amplification was observed only with T. saginata DNA(Fig. 8). Similarly, with 10 ng of DNA, amplification was pos-itive for T. saginata but negative for T. solium, T. taeniformis, E.granulossus, human, and bovine DNAs (Fig. 9).

Finally, the sensitivity of the PCR with HDP1-based primerswas determined with decreasing quantities of T. saginatagDNA as templates (Fig. 10). The PCR could detect 10 pg ofT. saginata DNA and yielded the characteristic ladder pattern,but a partial amplification could be observed even with as littleas 1 pg of T. saginata DNA.

Design of a T. saginata- and T. solium-specific multiplexPCR with HDP2-based primers. The HDP2-based primersPTs7S35F1, PTs7S35F2, and PTs7S35R1 were used to estab-lish a T. saginata- and T. solium-specific multiplex PCR. Theclearest results were obtained at an annealing temperature of56.5°C (data not shown). With 1 ng of gDNA from T. saginata,T. solium, T. taeniformis, E. granulossus, a human, and a calf, T.saginata, T. solium, and E. granulossus gDNAs yielded positivebut species-specific patterns for each of the three parasites(Fig. 11): two bands (of 600 and 170 bp) with T. saginata, oneband (of 170 bp) with T. solium, and two bands (of 900 and550) with E. granulosus. These data demonstrated the T. sagi-

nata species specificity of the PTs7S35F1-PTs7S35R1 primercombination on the 600-bp target sequence, as well as theT. saginata and T. solium specificity of the PTs7S35F2-PTs7S35R1 primer combination on the 150-bp target se-quence. Finally, the sensitivity of the multiplex PCR withHDP2-based primers was shown to be 10 pg of DNA when T.saginata, T. solium, and E. granulossus gDNAs were used astemplates (data not shown).

DISCUSSION

This paper describes the design and development of twoPCR tests for the specific and sensitive detection of T. solium,T. saginata, and E. granulosus. One PCR specific for T. saginatadetection uses primers based on the sequence of the publishedHDP1 T. saginata DNA fragment (19). The other is a multiplexPCR with primers derived from the sequence of another T.saginata DNA sequence, HDP2 (19), and which specificallyamplified T. saginata, T. solium, and E. granulosus DNAs. Thegenomic characteristics of each probe, their performance inthe PCR tests, and their potential applications are discussed.

The DNA sequences of HDP1 and HDP2 consisted of twoentirely distinct sequences of 1,272 and 3,954 bp, respectively.Stop codons were present at the beginning of each potentialreading frame (data not shown), and thus, there were no sig-nificant open reading frames that coded for proteins. No sim-ilarities were found between the HDP1 and HDP2 sequencesand any other sequence included in the EMBL and GenBankdatabases. The HDP1 fragment was composed of 53-bp mono-

FIG. 7. Demonstration of a unique T. saginata sequence (fragment 5HDP2)and shared T. saginata and T. solium sequences (fragment IPHDP2 and3PHDP2) within the T. saginata genomic sequence HDP2. Southern blotting wasdone with T. saginata (lanes 1) and T. solium (lanes 2) gDNAs (5 mg) cleavedwith the ClaI restriction enzyme. The digested gDNAs were probed with threenonoverlapping fragments derived from the HDP2 sequence fragments:5PHDP2 (A), IPHDP2 (B), and 3PHDP2 (C). The probes were labeled withdigoxigenin.

FIG. 8. Specificity of the PCR assay with HDP1-based primers and re-stricted T. saginata DNA. Samples of genomic DNA from T. saginata (A) inquantities of 40 ng (lane 1), 30 ng (lane 2), 20 ng (lane 3), and 10 ng (lane 4) andfrom T. solium (B) in quantities of 40 ng (lane 5), 30 ng (lane 6), and 20 ng (lane7) were amplified with the PTs4F1 and PTs4R1 primers. A negative non-DNAcontaining-control was also included (lane 8). The reactions were carried out asdescribed in Materials and Methods. The amplification products were fraction-ated on a 2% agarose gel and were stained with ethidium bromide. PromegaPCR molecular markers were used (lanes M).

FIG. 9. Specificity of the PCR assay with HDP1-based primers and re-stricted T. saginata DNA. Samples of genomic DNA (10 ng) from T. saginata(lane 1), T. solium (lane 2), T. taeniformis B (lane 3), T. taeniformis M (lane 4),E. granulosus (lane 5), a calf (lane 6), and a human (lane 7) were amplified withthe PTs4F1 and PTs4R1 primers as described in Materials and Methods. Anegative control without DNA was also included (lane 8). The amplificationproducts were fractionated on a 2% agarose gel and were stained with ethidiumbromide. Promega PCR molecular markers were used (lane M).

FIG. 10. Sensitivity of PCR amplification with HDP1-based primers. Sam-ples of genomic DNA of T. saginata, with input quantities of 10 ng (lane 1), 1 ng(lane 2), 100 pg (lane 3), 10 pg (lane 4), 1 pg (lane 5), 100 fg (lane 6), 10 fg (lane7), and 1 fg (lane 8) were amplified with the PTs4F1 and PTs4R1 primers for thePCR with HDP1-based primers. A negative control without DNA was alsoincluded (lane 9). The reactions were carried out as described in Materials andMethods. The amplification products were fractionated on a 2% agarose gel andwere stained with ethidium bromide. Promega PCR molecular markers wereused (lanes M).

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mers tandemly repeated 24 times, with a 55% A1T contentand with direct and inverse internal repeats in each monomer.An evaluation of the genomic organization by Southern blotanalysis, restriction enzyme mapping, and sequencing indi-cated that the 53-bp monomers were arranged in an estimated11,321 clustered tandem repeats along 23 kb or more of the T.saginata genome. The 53-bp monomer sequence was remark-ably conserved in comparison to other repetitive sequencesfrom parasite DNA that have been described (18, 42), withonly 0.3% divergence among the 24 sequenced units and withonly four base changes from the 53-bp consensus sequence.However, the degree of variation appeared to be increased atparticular sites along the HDP1 sequence. For example, the19th base of the monomer unit appeared to be a hot-spot site,and this mutation yielded a new ScaI recognition site, which inturn resulted in alteration of the consensus sequence, perhapsexplaining the Southern blot pattern obtained by ScaI diges-tion of T. saginata DNA and HDP1 probe hybridization (Fig.3). These observations suggested that the HDP1 sequence wassatellite DNA, and indeed, the 53-bp HDP1 monomer se-quence showed a high degree of similarity to satellite DNAs(22). Satellite DNA is defined as the DNA component whichrenatures rapidly in a eukaryotic genome, which consists ofshort sequences (5 to 200 bp) repeated many times in tandemin large clusters, and which is located in the heterochromaticregions of the chromosomes at both centromeric and telomericregions (6, 20, 22). Their abundance can vary from less than1% to more than 66% of the genome (38). Interestingly, threeof the HDP1 monomeric units had the same point mutation inthe same nucleotide and at the same position, suggesting thatthese mutations were not random variations. Possibly somemechanism analogous to similar previously described mecha-nisms acting on the satellite DNA was responsible (7, 18, 28,41, 42). Although the HDP1 genomic representation of 0.4%of T. saginata was very low for satellite DNA, a similarly lowpercentage had also been found in Caenorhabditis elegans sat-ellite DNA (21). In summary, therefore, we may conclude thatthe HDP1 sequence is satellite DNA that has repeats orga-nized in tandem arrays, that is characterized by a small unitsize and high copy number (22), and that perhaps has a struc-tural function, as has already been suggested (4, 18, 22, 30, 33,42). This is not the first time that highly repetitive DNAs, suchas satellite DNAs, which undergo rapid evolutionary changes,have been used as species-specific probes (17, 18, 38). Indeed,the specificity of the T. saginata 1,272-bp HDP1 target se-quence is exquisite, as Harrison et al. described before (19),

indicating that satellite DNA is, in general, species specific(38). However, there are few published data on these repetitiveelements in cestodes (5, 25, 31, 33).

The HDP2 probe with an A1T content of 45% was not arepetitive sequence. This fact and Southern blot analysis (Fig.4) suggested that HDP2 could be intergenic spacer DNA (22).Its dual specificity for DNAs of both T. saginata and T. soliumhas considerable practical potential, similar to the previousemployment of interspersed DNA fragments with large unitsizes and low to moderate copy numbers (10).

Taking into account the complete sequences and other char-acteristics of the two DNA probes (HDP1 specificity for T.saginata and HDP2 reactivity with both T. saginata and T.solium), primer sets were designed for the differential detec-tion of these two parasites by PCR. Thus, a T. saginata species-specific PCR with primers based on the sequence of the HDP1probe (19) and a multiplex PCR with primers based on thesequence of the HDP2 probe, which specifically amplified T.saginata, T. solium, and E. granulossus DNA sequences, weredeveloped.

The oligonucleotides designed from HDP1 provided a spe-cies-specific PCR amplification of T. saginata gDNA with acharacteristic ladder of 10 or 11 bands and with a differencebetween the bands of about 50 bp. This pattern suggested thatthe oligonucleotide primers hybridize to all the complementarysequences along the tandemly arranged repetitions in theHDP1 sequence. The PCR detected down to 10 pg of T. sagi-nata gDNA, and this high degree of sensitivity could be attrib-uted to the repetitive nature of the HDP1 sequence, as well asto the amplification power of the PCR. By calculating that oneTaenia sp. egg contains approximately 8 pg of gDNA (32), thePCR should be able to detect the gDNA from one T. saginataegg. Thus, the PCR with HDP1-based primers offers the pos-sibility of a sensitive, rapid, and specific method for the reliableidentification of T. saginata in the absence of a signal fromT. solium and other taeniids.

In order to achieve, in addition, a positive identification ofT. solium by PCR, a multiplex PCR was established by takingadvantage of both sequence specificity and the peculiar spec-ificity of the HDP2 probe. The test was based on T. saginatagenomic clone HDP2 and, moreover, distinguished T. saginata,T. solium, and E. granulosus through different amplificationpatterns, while it was negative for other taeniids. Specifically,these data demonstrated the T. saginata species specificity ofthe PTs7S35F1-PTs7S35R1 primer set and the T. saginata andT. solium specificity of the PTs7S35F2-PTs7S35R1 primer set.The exact nature of the specific products amplified from T.saginata, T. solium, and E. granulosus by primers PTs7S35F1,PTs7S35F2, and PTs7S35R1 remains to be determined andmay shed some light on the evolution of these organisms. Thesensitivity of the multiplex PCR was excellent, detecting aslittle as 10 pg of the taeniid gDNAs.

Both the PCR with HDP1-based primers and the multiplexPCR with HDP2-based primers are now ready for applicationto the differential detection of both T. saginata and T. solium inhumans and are clearly more efficient, specific, and sensitivethan previously reported Southern hybridization techniques (5,13, 19).

The most immediate priority is to distinguish T. saginata andT. solium infections in the clinical situation in order to rapidlyidentify human carriers of T. solium. Use of the PCR assays forthe positive identification of the parasites in dubious cysts,lesions, or cyst residues in domestic animals at the slaughter-house would aid in the appropriate treatment of the carcassesand in the control of these parasites in domestic livestock. Inthe future, the assays described in this paper could have a

FIG. 11. Differential detection of T. saginata, T. solium, and E. granulossus bymultiplex PCR. Samples of genomic DNA (1 ng) from T. saginata (lane 1), T.solium (lane 2), T. taeniformis B (lane 3), T. taeniformis M (lane 4), E. granulosus(lane 5), a calf (lane 6), and a human (lane 7) were amplified by the multiplexPCR based on the PTs7S35F1, PTs7S35F2, and PTs7S35R1 primers derivedfrom the T. saginata genomic sequence HDP2. A negative control without DNAwas also included (lane 8). The reactions were carried out as described inMaterials and Methods. The amplification products were fractionated on a 2%agarose gel and were stained with ethidium bromide. Promega PCR molecularmarkers were used (lanes M).

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major impact on epidemiological studies through the identifi-cation of tapeworm eggs in the environment, i.e., in watersupplies or on contaminated pasture, in addition to identifyinghuman tapeworm carriers. Importantly, in recent preliminaryexperiments we have been able to efficiently extract DNA fromtaeniid eggs. Finally, the sequences and primers used in thesestudies might also be used to determine the possible occur-rence of strains or geographical isolates of these parasites,perhaps via restriction enzyme polymorphism analyses of theamplified products as has been reported by McManus andcolleagues (3, 32).

ACKNOWLEDGMENTS

We thank J. M. Rubio for technical help in the design of the PCRwith HDP1-based primers and L. Benıtez and E. Rodrıguez for helpfuldiscussions.

This work was supported by grants from UE-INCO (grant DCIC18CT950002), FISS (grant 97/0141), and MEC/British Council (grantHB96-43).

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