Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR–RFLP and nested-PCR of ribosomal DNA ITS1 region

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<ul><li><p>ar</p><p>Cara</p><p>I, 28sidaCajaeda</p><p>a b s t r a c t</p><p>The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two larial</p><p>tivity of traditional serological methods to detect anti-larial anti-bodies are really poor. Furthermore, serological diagnosis oflariasis in immigrants from endemic areas is not appropriate asthese individuals usually have anti-larial antibodieswithout an ac-tive infection (Klion, 2008).</p><p>ucts with Ase I restriction enzyme yielded species-specic frag-ments that allowed the differential identication of the threelarial species. Additionally, an specic nested-PCR for each larialspecies was developed with primers based on their ITS1 regions toincrease the diagnosis sensitivity when poor or negative ITS1PCRproducts were observed. These molecular protocols were tested in40 blood samples from African adult immigrants attending in theHospital Insular of Gran Canaria, Canarias, Spain, for physicalexamination and routinely laboratory tests.</p><p>* Corresponding author. Fax: +34 91 5097034.** Corresponding author. Fax: +34 91 5097034.</p><p>Experimental Parasitology 127 (2011) 282286</p><p>Contents lists availab</p><p>l</p><p>.eE-mail addresses: mjimenez@isciii.es (M. Jimnez), tgarate@isciii.es (T. Grate).1. Introduction</p><p>Human lariases are restricted to the tropics and subtropicsendemic areas where more than 120 million people are estimatedto be infected (WHO, 2010). In the last years, there have been an in-crease of African immigrants to Spain from lariasis endemic areaswhere Loa loa, Mansonella perstans and Wuchereria bancrofti areco-endemic (Carrillo et al., 2004; Pardo et al., 2006). Thesedemographic changes require for a better lariae species-specicmolecular diagnosis tests to solve problems derived from themicro-scopical identication of these microlariae in patients blood thatneeds a great expertise and is time consuming (Walther andMuller,2003; Nuchprayoon, 2009). In addition, both specicity and sensi-</p><p>In a previous study, Nuchprayoon et al. (2005) reported on anassay system that uses a polymerase chain reactionrestrictionfragment length polymorphism (PCRRFLP), targeted the inter-nally-transcribed spacer 1 (ITS1) region of the ribosomal RNA gene,based on the Ase I restriction enzyme digestion that discriminatesbetween ve species of larial nematodes:W. bancrofti, Brugia ma-layi, Brugia pahangi, Dirolaria inmitis and Dirolaria repens, sug-gesting the utility of this PCR in the differential detection ofother lariae as L. loa and M. perstans that sympatrically co-existin West and Central Africa.</p><p>Thus, the objective of the present work was to develop and toevaluate this PCRRFLP targeted the ITS1 from L. loa, M. perstansand W. bancrofti. The subsequent digestion of amplication prod-a r t i c l e i n f o</p><p>Article history:Received 22 March 2010Received in revised form 4 June 2010Accepted 15 June 2010Available online 19 June 2010</p><p>Keywords:Loa loaMansonella perstansWuchereria bancroftiITS1PCRRFLPNested-PCRDifferential detection0014-4894/$ - see front matter 2010 Elsevier Inc. Adoi:10.1016/j.exppara.2010.06.019nematodes, Loa loa and Mansonella perstans, was amplied and further sequenced to develop an spe-cies-specic polymerase chain reactionrestriction fragment length polymorphism (PCRRFLP) protocolfor the differentiation of both species from Wuchereria bancrofti, three larial nematodes with blood cir-culating microlariae. The ITS1PCR product digested with the restriction endonuclease Ase I generatedan specic diagnostic pattern for each of the three species. Moreover, three new specic nested-PCRs, tar-geting the ITS1 region, for differential detection of L. loa,M. perstans andW. bancrofti were developed andused when the ITS1PCR products were insufcient for the Ase I enzymatic digestion. These larial spe-cies-specic molecular protocols were evaluated in forty blood samples from African adult immigrantsattending in the Hospital Insular of Gran Canaria, Canarias, Spain.</p><p> 2010 Elsevier Inc. All rights reserved.Research Brief</p><p>Detection and discrimination of Loa loa, Mbancrofti by PCRRFLP and nested-PCR of</p><p>Maribel Jimnez a,*, Luis Miguel Gonzlez a, CristinaAntonio Muro d, Jos Luis Prez-Arellano b, Teresa Ga Servicio de Parasitologa, Centro Nacional de Microbiologa, Instituto de Salud Carlos IIbDepartamento Ciencias Mdicas y Quirrgicas, Facultad de Ciencias de la Salud, UnivercUnidad de Medicina Tropical, Servicio de Enfermedades Infecciosas, Hospital Ramn yd Laboratorio de Inmunologa Parasitaria y Molecular, Centro de Investigacin de Enferm37007 Salamanca, Spain</p><p>Experimenta</p><p>journal homepage: wwwll rights reserved.nsonella perstans and Wuchereriaibosomal DNA ITS1 region</p><p>rranza b, Begoa Bailo a, Ana Prez-Ayala c,te a,**</p><p>220 Majadahonda, Madrid, Spaind de Las Palmas de Gran Canaria, 35080 Las Palmas de Gran Canaria, Spainl, Madrid, Spaindes Tropicales de la Universidad de Salamanca, Campus Miguel de Unamuno s/n,</p><p>le at ScienceDirect</p><p>Parasitology</p><p>lsevier .com/locate /yexpr</p></li><li><p>2. Materials and methods</p><p>2.1. Blood samples and genomic DNA extraction</p><p>Peripheral blood samples were collected from 40 adult immi-grants of different African geographic origin attending in the Hos-pital Insular of Gran Canaria, Canarias, Spain. The Hospital Insularof Gran Canaria Ethics Review Committee approved the protocolsfor obtaining blood samples from patients enrolled in the presentstudy. After their written consent for parasitological diagnosis,Knotts test for microlariae identication was carried out (Knott,1939) (Table 1).</p><p>Whole blood with EDTA (n = 28) and blood in Whatman paper(n = 12) was used for DNA extraction. DNA was puried using theQIAamp DNA Blood Extraction Kit (Qiagen, Hilden, Germany)according to the manufacturers instructions. Quantication andpurity of the DNA samples was determined by spectrophotometrywith a NanoDrop ND-1000 spectrophotometer (Nucliber, Madrid,Spain), and the samples stored at 20 C until use.</p><p>2.2. PCR amplication of ITS1 from L. loa and M. perstans</p><p>Genomic DNA obtained from samples No. 4 and No. 10 (Table 1)was used to amplify the ITS1 region of the ribosomal RNA genefrom L. loa and M. perstans, respectively, as previously describedby Nuchprayoon et al. (2005) with slight modications. Universal</p><p>primers ITS1-F (50-GGTGAACCTGCGGAAGGATC-30) and ITS1-R (50-CTCAATGCGTCTGCAATTCGC-30) situated in conserved regions of18S and 5.8S subunits of ribosomal RNA gene were used (Fig. 1).Individual PCRs were carried out in a nal volume of 50 ll. Theamount of DNA in each reaction was 200 ng. The master mix forboth PCRs consisted of 5 ll of 10 PCR buffer for Ampli-Taq Goldpolymerase (Applied Biosystem, UK), 1 ll Ampli-Taq-Gold poly-merase (5 U/ll) (Applied Biosystem), 0.2 mM of each deoxynucle-oside triphosphate (dNTPs) (Amersham Pharmacia Biotech,Sweden), 1.68 mM of MgCl2 (Applied Biosystem), 4 ll of BSADNAse Free (0.8 lg/ll) (Amersham Pharmacia Biotech) and200 ng/ll of each primer ITS1-F and ITS1-R. Working conditionswere 1 cycle 95 C 9 min then 35 cycles (94 C 30 s, 58 C 30 s,72 C 45 s) followed by 1 cycle 72 C for 10 min. The PCR productswere separated on ethidium bromide 2% agarose gel (Conda,Spain), visualized under UV light and photographed.</p><p>2.3. Cloning and sequence analysis of the ITS1PCR products</p><p>The ITS1PCR products obtained from L. loa and M. perstanswere removed from the gel under UV exposure and puried by aQIAquick Gel Extraction Kit (Qiagen). Afterwards, the samples weresubcloned into the plasmid PCR4-TOPO (Invitrogen, UK) andthree of the clones sequenced two times each with ABI 3700DNA sequencer (Applied Biosystems). Nucleotide sequences ob-tained for M. perstans and L. loa were aligned with the W. bancrofti</p><p>Table 1Results obtained by microscopic diagnosis and the different PCR methods used in the study.</p><p>No. Sample type Geographical origin Knott technique ITS1PCR ITS1RFLP Nested-ITS1</p><p>1 Wp Conakry Guinea M. perstans Positive n.d. M. perstans2 Wp Mali M. perstans Negative n.d. M. perstans3 Wp Cameroon M. perstans Negative n.d. M. perstans4 Wp Cameroon L. loa Positive L. loa n.d.5 Wp Cameroon M. perstans Positive M. perstans n.d.6 Wp Ghana M. perstans Positive M. perstans n.d.</p><p>ans</p><p>15 Blood Mauritania Negative</p><p>Negative n.d. W. bancroftiNegative n.d. Negative</p><p>M. Jimnez et al. / Experimental Parasitology 127 (2011) 282286 28316 Wp Mali M. perstans17 Wp Sierra Leone M. perstans18 Wp Sierra Leone W. bancrofti19 Wp Equatorial Guinea Negative20 Wp Equatorial Guinea M. perstans21 Blood Guinea Bissau M. perstans22 Blood Guinea Bissau M. perstans23 Blood Equatorial Guinea M. perstans24 Blood Equatorial Guinea M. perstans25 Blood Equatorial Guinea M. perstans26 Blood Nigeria M. perstans27 Blood Morocco Negative28 Blood Morocco Negative29 Blood Morocco Negative30 Blood Equatorial Guinea Negative31 Blood Equatorial Guinea Negative32 Blood Nigeria M. perstans33 Blood Equatorial Guinea Negative34 Blood Mali Negative35 Blood Equatorial Guinea Negative36 Blood Equatorial Guinea Negative37 Blood Morocco Negative38 Blood Morocco Negative39 Blood Morocco Negative7 Wp Equatorial Guinea L. loa8 Blood Equatorial Guinea L. loa/ M. perstans9 Blood Guinea Bissau W. bancrofti/ M. perst</p><p>10 Blood Guinea Bissau M. perstans11 Blood Aiun Negative12 Blood Morocco Negative13 Blood Mauritania Negative14 Blood Mauritania Negative40 Blood Morocco Negative</p><p>Wp, Whatman paper; n.d., not done.Negative n.d. M. perstansPositive M. perstans n.d.Positive M. perstans n.d.Positive M. perstans n.d.Positive M. perstans n.d.Positive M. perstans n.d.Positive M. perstans n.d.Negative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativePositive M. perstans n.d.Negative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativePositive n.d. L. loaPositive L. loa/ M. perstans L. loa / M. perstansPositive W. bancrofti/ M. perstans W. bancrofti/M. perstansPositive M. perstans n.d.Negative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. NegativeNegative n.d. M. perstansPositive M. perstans n.d.Negative n.d. Negative</p></li><li><p>differential species-specic diagnosis of L. loa, M. perstans and W.</p><p>7),ved1) aedto</p><p>Par482 bp sequence (GenBank Accession No. AY621473). Multiplesequence alignments were made using ClustalW method. The pre-dicted Ase I restriction sites were determined with the DNASTARLasergene v7.1 program.</p><p>2.4. Polymerase chain reactionrestriction fragment lengthpolymorphism</p><p>The ITS1PCR protocol was applied to genomic DNA obtainedfrom the 40 samples, as described above. After amplication,25 ll of each PCR sample were electrophoresed on ethidium bro-mide 2% agarose D5 (Conda) gel in 1 TAE buffer, visualized underUV light and photographed.</p><p>Each sample was tested twice with the primers sets (see above).</p><p>Fig. 1. Alignment of the ITS1 from L. loa (457 bp; GenBank Accession No. DQ99549GenBank Accession No. AY621473). Primers ITS1-F and ITS1-R are located in conser2005). For the nested-PCRs specic forward primers for L. loa (LlF1), M. perstans (MpF(LlR1) designed into the ITS1 region and reverse primer forW. bancrofti (WbR1) designM. perstans (MpR1) was designed into the ITS1 region. Underlined bases correspond</p><p>284 M. Jimnez et al. / ExperimentalNegative controls without DNA were included in each PCR. Subse-quently, 25 ll of each PCR product were digested with 5 U of Ase Iat 37 C in 50 ll of reaction volume according to themanufacturersprotocols (New England Biolabs, UK). The digested PCR productswere then fractionated in 3% agarose gel (Conda), visualized underUV light, after ethidium bromide staining, and photographed.</p><p>2.5. Primer design and nested-PCRs</p><p>For the newnested-PCRs, specic forwardprimers for L. loa (LlF1:50-GATGATGATATATGATGAAG-30), M. perstans (MpF1: 50-CAATGAAATGTTATCCATA-30) and W. bancrofti (WbF1: 50-GTGTTACTAATATAGATTG-30) were designed to encompass the ITS1 region. Reverseprimer for L. loa (LlR1: 50-TTAAGCTATCGCTTTATCTTC-30) was alsodesigned into the ITS1 region. Reverse primer for W. bancrofti(WbR1: 50-GCTGCGTTCTTCATCGATCCACGAGCC-30) was designedinto the 5.8S region. Both reverse primers were common in all thethree species. Specic reverse primer for M. perstans (MpR1: 50-AAATGCTTATTAAGTCTACTTAATTAAT-30) was designed into the ITS1region (Fig. 1). The nested-PCRs were carried out with 1 ll of DNAtemplate from the rst PCR reaction (ITS1PCR protocol), diluted4:1000 in DNase-free water (Promega). The working conditionswere 1 cycle 95 C 9 min then 35 cycles (94 C 30 s, 58 C 30 s,72 C 45 s) followed by 1 cycle 72 C for 10 min. The nested-PCRssamples were electrophoresed through a 2% agarose D1 (Conda)gel in 1 TAE buffer and visualized by trans-illumination withultra-violet light. Each sample was tested twice with each set ofbancrofti, based on the ITS1 region. The ITS1 regions of L. loa andM. perstans were amplied and their sequences compared to theW. bancrofti ITS1. Thus, PCR products were visualized and foundto be 457 bp for L. loa, 484 bp forM. perstans and 482 bp forW. ban-crofti (Fig. 2). The 457 bp-ITS1 from L. loa and the 484 bp-ITS1 fromprimers. Negative controls without DNA were included in eachPCR. To avoid PCR contamination, sample preparation, reactionsset-up and PCR amplications were carried out in separate rooms,with different lab coats and gloves.</p><p>3. Results and discussion</p><p>The present study reports a PCRRFLP and three nested-PCRs for</p><p>M. perstans (484 bp; GenBank Accession No. DQ995498) and W. bancrofti (482 bp;regions of 18S and 5.8S ribosomal DNA subunits, respectively (Nuchprayoon et al.,ndW. bancrofti (WbF1) were designed into the ITS1 region. Reverse primer for L. loainto the 5.8S region were common in all the three species. Specic reverse primer forthe primers. Predicted Ase I restriction sites are indicated in boxes.</p><p>asitology 127 (2011) 282286M. perstans were deposited in the GenBank (Accession Nos.DQ995497 and Q995498, respectively). These sequences togetherwith the W. bancrofti 482 bp sequence, GenBank Accession No.AY621473, were aligned by ClustalW Multiple alignment (Fig. 1).Analysis of each individual sequence data provided the predictedAse I restriction endonuclease pattern, with an specic diagnosispattern for each of the three larial species (Table 2). In addition,the study of the multiple alignments allowed us to design threedifferent set of primers and used them in three specic and inde-pendent nested-PCR protocols to distinguish the larial species(Fig. 1), more efciently than the PCRRFLP system describedabove.</p><p>To evaluate the ITS1RFLP as a differential diagnosis method inthe specic identication of the three larial species, the ITS1RFLP was carried out in genomic DNA obtained from 40 blood sam-ples collected from adult immigrants of different African geo-graphic origin (Table 1). The PCR products were visualized andfound to be 457 bp for L. loa, 484 bp for M. perstans and 482 bpfor W. bancrofti (Fig. 2A, Table 2). Digestion of the ITS1PCR prod-ucts yielded an exclusive spec...</p></li></ul>

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