differential diagnosis of taenia saginata and taenia solium

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

    Feb. 2000, p. 737744 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 80C for 10 min, followed byneutralization with 0.25 M Tris-HCl (pH 7.5)0.25 M HCl12.53 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 manufacturers 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 manufacturers instructions. Hybridizations were conducted overnightunder high-stringency conditions at 68C. After hybridization, the filters werewashed at 68C for 10 min in 23 SSC0.1% sodium dodecyl sulfate (SDS) andthen for a further 40 min in 0.13 SSC0.1% 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 manufacturers 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 94C for 5 min (initial denaturation),followed by 35 cycles at 94C for 1 min, 60C for 30 s, 72C for 30 s, and 72C for10 min (final extens

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