412 lines
41 KiB
XML
412 lines
41 KiB
XML
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<mods:title id="B069A17886E859D480F34055A9C3D491">Molecular screening of tsetse flies and cattle reveal different Trypanosoma species including T. grayi and T. theileri in northern Cameroon</mods:title>
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<mods:namePart id="8A2AC125E3D18708480C9891073F5DBD">Ngomtcho, Sen Claudcne Henlcette</mods:namePart>
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<mods:namePart id="5FEEC9981CF563424E370D6854E37A4A">Webel, Judcth Sophce</mods:namePart>
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<mods:namePart id="D49C5BB420075074D7470DB7A7538DB5">Bum, Elcmabeth Ngo</mods:namePart>
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<mods:namePart id="53E7CC988F042A001AD0B4C3EF053EA5">Gbem, Thaddeum Tellumun</mods:namePart>
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<mods:namePart id="D4C1B232BC5F9E9BB2BE199F1B473C45">Kelm, SØlge</mods:namePart>
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<mods:namePart id="C3F10FE091058EE8D39BCFF2830FFC66">Achukwc, Mbunkah Dancel</mods:namePart>
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<mods:title id="8053F666DC75AAFAB7F5FE041124F331">Parasites & Vectors</mods:title>
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<mods:date id="E7B90A6283CCF4AB1E2415F0BCC3C6EA">2017</mods:date>
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<mods:title id="DD020B80CBA7E4965E8568786E3C35A1">631</mods:title>
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<mods:number id="FDC64FA97AAAB6339DB33CDA6B8DD20A">2017-12-29</mods:number>
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<mods:identifier id="A3FC215542C041490E3CB7DF4597ECA6" type="DOI">10.1186/s13071-017-2540-7</mods:identifier>
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<mods:identifier id="68AFA80688E1D1061FCC27F0BE429F8B" type="GBIF-Dataset">26cf0558-8e86-4249-ad88-713c4c392f12</mods:identifier>
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<mods:identifier id="C81B080A5EFC25ACB8462DAD38356EE4" type="ISSN">1756-3305</mods:identifier>
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<mods:identifier id="1A0EC1A9DAACC2B52AA1CE63CF030A68" type="PMC">PMC5747950</mods:identifier>
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<treatment id="03DA9B06FF84B46AD63EFF64A04AF92A" ID-DOI="http://doi.org/10.5281/zenodo.11645809" ID-GBIF-Taxon="232750360" ID-Zenodo-Dep="11645809" LSID="urn:lsid:plazi:treatment:03DA9B06FF84B46AD63EFF64A04AF92A" httpUri="http://treatment.plazi.org/id/03DA9B06FF84B46AD63EFF64A04AF92A" lastPageId="4" lastPageNumber="5" pageId="3" pageNumber="4">
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<subSubSection id="C369799BFF84B46DD63EFF64A2FDFE8B" pageId="3" pageNumber="4" type="nomenclature">
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<paragraph id="8BCC2A10FF84B46DD63EFF64A2FDFE8B" blockId="3.[813,1437,238,709]" pageId="3" pageNumber="4">
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<heading id="D0849D7CFF84B46DD63EFF64A2FDFE8B" bold="true" fontSize="9" level="2" pageId="3" pageNumber="4" reason="4">
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<emphasis id="B907F602FF84B46DD63EFF64A2FDFE8B" bold="true" pageId="3" pageNumber="4">
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Identification of
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<taxonomicName id="4C735193FF84B46DD6CEFF64A550FEAB" box="[989,1076,238,260]" pageId="3" pageNumber="4" rank="subTribe" subTribe="Glossina">
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<emphasis id="B907F602FF84B46DD6CEFF64A550FEAB" bold="true" box="[989,1076,238,260]" italics="true" pageId="3" pageNumber="4">Glossina</emphasis>
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</taxonomicName>
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species by sequencing of the
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<emphasis id="B907F602FF84B46DD63EFE84A234FE8B" bold="true" box="[813,848,270,292]" italics="true" pageId="3" pageNumber="4">cox</emphasis>
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1 gene
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</emphasis>
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</heading>
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</paragraph>
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</subSubSection>
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<subSubSection id="C369799BFF84B46AD63EFEA7A04AF92A" lastPageId="4" lastPageNumber="5" pageId="3" pageNumber="4" type="description">
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<paragraph id="8BCC2A10FF84B46DD63EFEA7A500FD6A" blockId="3.[813,1437,238,709]" pageId="3" pageNumber="4">
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To cdentcfy tsetse fly specces also on the genetcc level, we amplcfced and sequenced part of the
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<taxonomicName id="4C735193FF84B46DD1AEFEC7A584FECB" box="[1213,1248,333,356]" class="Insecta" family="Glossinidae" genus="Glossina" kingdom="Animalia" order="Diptera" pageId="3" pageNumber="4" phylum="Arthropoda" rank="species" species="cox">
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<emphasis id="B907F602FF84B46DD1AEFEC7A584FECB" box="[1213,1248,333,356]" italics="true" pageId="3" pageNumber="4">cox</emphasis>
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</taxonomicName>
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1 gene [
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<bibRefCitation id="EFE257E1FF84B46DD025FEC7A437FECA" author="Dsel NA & Lawton SP & Ravel S & Choc KS & Lehane MJ & Robcnmon AS" box="[1334,1363,333,357]" pageId="3" pageNumber="4" pagination="227 - 39" refId="ref12585" refString="28. Dsel NA, Lawton SP, Ravel S, Choc KS, Lehane MJ, Robcnmon AS, et al. Moleculal phslogenetccm of tmetme flcem (Dcptela: Glommcncdae) bamed on mctochondlcal (COI, 16 S, ND 2) and nucleal lcbomomal DNA mequencem, wcth an emphamcm on the palpalis gloup. Mol Phslogenet Evol. 2008; 49: 227 - 39." type="journal article" year="2008">28</bibRefCitation>
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] uscng the prcmers lcsted cn
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<tableCitation id="C6F11FABFF84B46DD106FEE7A502FE2A" box="[1045,1126,365,389]" captionStart="Table 1" captionStartId="3.[151,210,1246,1267]" captionTargetPageId="3" captionText="Table 1 Plcmelm umed cn thcm mtuds to amplcfs dcffelent Trypanosoma mpeccem" httpUri="http://table.plazi.org/id/DF0C7A98FF84B46DD584FB54A21CFB5C" pageId="3" pageNumber="4" tableUuid="DF0C7A98FF84B46DD584FB54A21CFB5C">Table 1</tableCitation>
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.
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reactcons (25 μl) contacned 5 μl template DNA, 2 μM of prcmers, 20 μM of dNTPs (Thermo Fcsher Sccentcfcc, Drececch,
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<collectingCountry id="F3646A80FF84B46DD03CFE27A4F1FE6A" box="[1327,1429,429,453]" name="Germany" pageId="3" pageNumber="4">Germany</collectingCountry>
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) and Dream
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<emphasis id="B907F602FF84B46DD6B4FE47A2B4FE4B" box="[935,976,461,484]" italics="true" pageId="3" pageNumber="4">Taq</emphasis>
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||
Green polymerase (Thermo Fcsher Sccentcfcc).
|
||
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cyclcng reactcons cncluded an cnctcal denaturatcon 95 °C for 5 mcn, followed by 35 cycles of 1 mcn at 94 °C, 1 mcn at 55 °C, and 2 mcn at 72 °C, and a fcnal elongatcon of 10 mcn at 72 °C. Fragments were purcfced on a 1.5% agarose gel contacncng 0.5 μg/ml of
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<collectionCode id="ED62B2D5FF84B46DD05DFDE7A4F9FD2A" box="[1358,1437,621,645]" pageId="3" pageNumber="4">SERVA</collectionCode>
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DNA Stacn G (
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<collectionCode id="ED62B2D5FF84B46DD6CCFD07A556FD0A" box="[991,1074,653,677]" pageId="3" pageNumber="4">SERVA</collectionCode>
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, Hecdelberg,
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<collectingCountry id="F3646A80FF84B46DD1DEFD07A457FD0A" box="[1229,1331,653,677]" name="Germany" pageId="3" pageNumber="4">Germany</collectingCountry>
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) and sequenced as descrcbed below.
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</paragraph>
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<paragraph id="8BCC2A10FF84B46DD63EFD64A2F0FB8A" blockId="3.[813,1437,750,1189]" pageId="3" pageNumber="4">
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<emphasis id="B907F602FF84B46DD63EFD64A439FCAB" bold="true" box="[813,1373,750,772]" pageId="3" pageNumber="4">
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Detection and identification of
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<taxonomicName id="4C735193FF84B46DD16FFD64A46DFCAB" box="[1148,1289,750,772]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="genus">
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<emphasis id="B907F602FF84B46DD16FFD64A46DFCAB" bold="true" box="[1148,1289,750,772]" italics="true" pageId="3" pageNumber="4">Trypanosoma</emphasis>
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</taxonomicName>
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species
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</emphasis>
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Nested
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<collectionCode id="ED62B2D5FF84B46DD697FC87A2D1FC8A" box="[900,949,781,805]" pageId="3" pageNumber="4">PCR</collectionCode>
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targetcng the cnternal transcrcbed spacer 1 (
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<specimenCode id="DBD5826BFF84B46DD626FCA7A20EFCEA" box="[821,874,813,837]" collectionCode="ITS" pageId="3" pageNumber="4">ITS1</specimenCode>
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) regcon of the trypanosome rcbosomal DNA, whcch separates 28S from
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<geoCoordinate id="EE474CD7FF84B46DD174FCC7A5F0FCCA" box="[1127,1172,845,869]" degrees="5.8" direction="south" orientation="latitude" pageId="3" pageNumber="4" precision="5555" value="-5.8">5.8S</geoCoordinate>
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RNA, was performed. Fcrst, cdentcfccatcon was made uscng scze estcmatcon of amplccons generated by use of genercc prcmers (
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<tableCitation id="C6F11FABFF84B46DD02FFC07A4E9FC0A" box="[1340,1421,909,933]" captionStart="Table 1" captionStartId="3.[151,210,1246,1267]" captionTargetPageId="3" captionText="Table 1 Plcmelm umed cn thcm mtuds to amplcfs dcffelent Trypanosoma mpeccem" httpUri="http://table.plazi.org/id/DF0C7A98FF84B46DD584FB54A21CFB5C" pageId="3" pageNumber="4" tableUuid="DF0C7A98FF84B46DD584FB54A21CFB5C">Table 1</tableCitation>
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). For further confcrmatcon of the specces, prcmer sets speccfcc for several
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<taxonomicName id="4C735193FF84B46DD6F7FC47A51DFC4B" box="[996,1145,973,996]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="genus">
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||
<emphasis id="B907F602FF84B46DD6F7FC47A51DFC4B" box="[996,1145,973,996]" italics="true" pageId="3" pageNumber="4">Trypanosoma</emphasis>
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</taxonomicName>
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specces were descgned to amplcfy regcons of trypanosomal 18S rcbosomal RNA (
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<tableCitation id="C6F11FABFF84B46DD627FB87A2E0FB8A" box="[820,900,1037,1061]" captionStart="Table 1" captionStartId="3.[151,210,1246,1267]" captionTargetPageId="3" captionText="Table 1 Plcmelm umed cn thcm mtuds to amplcfs dcffelent Trypanosoma mpeccem" httpUri="http://table.plazi.org/id/DF0C7A98FF84B46DD584FB54A21CFB5C" pageId="3" pageNumber="4" tableUuid="DF0C7A98FF84B46DD584FB54A21CFB5C">Table 1</tableCitation>
|
||
).
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||
</paragraph>
|
||
<paragraph id="8BCC2A10FF84B46AD651FBA7A378FDCA" blockId="3.[813,1437,750,1189]" lastBlockId="4.[151,776,236,933]" lastPageId="4" lastPageNumber="5" pageId="3" pageNumber="4">
|
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<collectionCode id="ED62B2D5FF84B46DD651FBA7A20AFBEA" box="[834,878,1069,1093]" pageId="3" pageNumber="4">ITS</collectionCode>
|
||
1 nested
|
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<collectionCode id="ED62B2D5FF84B46DD6DCFBA7A564FBEA" box="[975,1024,1069,1093]" pageId="3" pageNumber="4">PCR</collectionCode>
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||
reactcons for detectcon of trypanosomal DNA wcth genercc prcmers were performed cn a 25 μl reactcon volume contacncng Dream
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<emphasis id="B907F602FF84B46DD00DFBE7A423FB2B" box="[1310,1351,1133,1156]" italics="true" pageId="3" pageNumber="4">Taq</emphasis>
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Green DNA polymerase and Dream
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||
<emphasis id="B907F602FF84B46DD162FB07A5FEFB0B" box="[1137,1178,1165,1188]" italics="true" pageId="3" pageNumber="4">Taq</emphasis>
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Green buffer (Thermo Sccentcfcc). The fcrst reactcon contacncng
|
||
<quantity id="4C8B87F5FF83B46AD744FF67A3EDFEAA" box="[599,649,237,261]" metricMagnitude="-12" metricUnit="kg" metricValue="1.0" pageId="4" pageNumber="5" unit="ng" value="1.0">1 ng</quantity>
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||
/μl of DNA template and 2 μM of prcmers (
|
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<collectionCode id="ED62B2D5FF83B46AD703FE87A35EFE8A" box="[528,570,269,293]" pageId="4" pageNumber="5">ITS</collectionCode>
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||
1-OutF and
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||
<collectionCode id="ED62B2D5FF83B46AD7DAFE87A392FE8A" box="[713,758,269,293]" pageId="4" pageNumber="5">ITS</collectionCode>
|
||
1- OutR,
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||
<tableCitation id="C6F11FABFF83B46AD5F1FEA7A056FEEA" box="[226,306,301,325]" captionStart="Table 1" captionStartId="3.[151,210,1246,1267]" captionTargetPageId="3" captionText="Table 1 Plcmelm umed cn thcm mtuds to amplcfs dcffelent Trypanosoma mpeccem" httpUri="http://table.plazi.org/id/DF0C7A98FF84B46DD584FB54A21CFB5C" pageId="4" pageNumber="5" tableUuid="DF0C7A98FF84B46DD584FB54A21CFB5C">Table 1</tableCitation>
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||
) was run under the followcng condctcons: cnctcal denaturatcon at 95 °C for 1 mcn, 30 cycles of 94 °C for 1 mcn, annealcng at 54 °C for 30 s, elongatcon at 72 ° C for 30 s, followed by a fcnal elongatcon step at 72 °C for 5 mcn. Fcrst
|
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<collectionCode id="ED62B2D5FF83B46AD45DFE27A01BFE6A" box="[334,383,429,453]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
products were dcluted 80-fold and 1 μl of thcs dclutcon was used for the second
|
||
<collectionCode id="ED62B2D5FF83B46AD785FE47A3A3FE4A" box="[662,711,461,485]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
reactcon wcth
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||
<collectionCode id="ED62B2D5FF83B46AD407FE67A05AFDAA" box="[276,318,493,517]" pageId="4" pageNumber="5">ITS</collectionCode>
|
||
1-InF and
|
||
<collectionCode id="ED62B2D5FF83B46AD4D2FE67A08FFDAA" box="[449,491,493,517]" pageId="4" pageNumber="5">ITS</collectionCode>
|
||
1-InR prcmers (
|
||
<tableCitation id="C6F11FABFF83B46AD7B7FE67A398FDAA" box="[676,764,493,517]" captionStart="Table 1" captionStartId="3.[151,210,1246,1267]" captionTargetPageId="3" captionText="Table 1 Plcmelm umed cn thcm mtuds to amplcfs dcffelent Trypanosoma mpeccem" httpUri="http://table.plazi.org/id/DF0C7A98FF84B46DD584FB54A21CFB5C" pageId="4" pageNumber="5" tableUuid="DF0C7A98FF84B46DD584FB54A21CFB5C">Table 1</tableCitation>
|
||
) under the same condctcons as the fcrst reactcon. As dcscussed by Adams et al. [
|
||
<bibRefCitation id="EFE257E1FF83B46AD4B0FDA7A0A5FDEA" author="Adamm ER & Malele II & Mmangc AR & Gcbmon WC" box="[419,449,557,581]" pageId="4" pageNumber="5" pagination="103 - 9" refId="ref12412" refString="24. Adamm ER, Malele II, Mmangc AR, Gcbmon WC. Tlspanomome cdentcfccatcon cn wcld tmetme populatconm cn Tanzanca umcng genelcc plcmelm to amplcfs the lcbomomal RNA ITS 1 legcon. Acta Tlop. 2006; 100: 103 - 9." type="journal article" year="2006">24</bibRefCitation>
|
||
], wcth thcs nested
|
||
<collectionCode id="ED62B2D5FF83B46AD79EFDA7A3A4FDEA" box="[653,704,557,581]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
, DNA of a scngle parascte can be detected.
|
||
</paragraph>
|
||
<caption id="DF0C7A98FF84B46DD584FB54A21CFB5C" ID-Table-UUID="DF0C7A98FF84B46DD584FB54A21CFB5C" box="[151,888,1246,1267]" httpUri="http://table.plazi.org/id/DF0C7A98FF84B46DD584FB54A21CFB5C" pageId="3" pageNumber="4" startId="3.[151,210,1246,1267]" targetBox="[151,1436,1283,1897]" targetIsTable="true" targetPageId="3" targetType="table">
|
||
<paragraph id="8BCC2A10FF84B46DD584FB54A21CFB5C" blockId="3.[151,888,1246,1267]" box="[151,888,1246,1267]" pageId="3" pageNumber="4">
|
||
<emphasis id="B907F602FF84B46DD584FB54A182FB5C" bold="true" box="[151,230,1246,1267]" pageId="3" pageNumber="4">Table 1</emphasis>
|
||
Plcmelm umed cn thcm mtuds to amplcfs dcffelent
|
||
<taxonomicName id="4C735193FF84B46DD7BEFB54A24FFB5C" box="[685,811,1246,1267]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="genus">
|
||
<emphasis id="B907F602FF84B46DD7BEFB54A24FFB5C" box="[685,811,1246,1267]" italics="true" pageId="3" pageNumber="4">Trypanosoma</emphasis>
|
||
</taxonomicName>
|
||
mpeccem
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8BCC2A10FF84B46DD584FA89A303F8C6" pageId="3" pageNumber="4">
|
||
<table id="F973D8B0FF844B91D584FA89A4F8F8C6" box="[151,1436,1283,1897]" gridcols="6" gridrows="17" pageId="3" pageNumber="4">
|
||
<tr id="35432852FF844B91D584FA89A4F8FAB9" box="[151,1436,1283,1302]" gridrow="0" pageId="3" pageNumber="4">
|
||
<th id="7692412EFF844B91D584FA89A19DFAB9" box="[151,249,1283,1302]" gridcol="0" gridrow="0" pageId="3" pageNumber="4">Name</th>
|
||
<th id="7692412EFF844B91D42FFA89A303FAB9" box="[316,615,1283,1302]" gridcol="1" gridrow="0" pageId="3" pageNumber="4">
|
||
Sequence (5
|
||
<emphasis id="B907F602FF84B46DD4B6FA89A0D7FAB9" box="[421,435,1283,1302]" italics="true" pageId="3" pageNumber="4">′–</emphasis>
|
||
3
|
||
<emphasis id="B907F602FF84B46DD4ADFA89A0A6FAB9" box="[446,450,1283,1302]" italics="true" pageId="3" pageNumber="4">′</emphasis>
|
||
)
|
||
</th>
|
||
<th id="7692412EFF844B91D7BBFA89A387FAB9" box="[680,739,1283,1302]" gridcol="2" gridrow="0" pageId="3" pageNumber="4">TA (°C)</th>
|
||
<th id="7692412EFF844B91D636FA89A296FAB9" box="[805,1010,1283,1302]" gridcol="3" gridrow="0" pageId="3" pageNumber="4">Amplccon mcze (bp)</th>
|
||
<th id="7692412EFF844B91D126FA89A460FAB9" box="[1077,1284,1283,1302]" gridcol="4" gridrow="0" pageId="3" pageNumber="4">Speccem</th>
|
||
<th id="7692412EFF844B91D055FA89A4F8FAB9" box="[1350,1436,1283,1302]" gridcol="5" gridrow="0" pageId="3" pageNumber="4">Refelence</th>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584FAACA4F8FA96" box="[151,1436,1318,1337]" gridrow="1" pageId="3" pageNumber="4">
|
||
<th id="7692412EFF844B91D584FAACA19DFA96" box="[151,249,1318,1337]" gridcol="0" gridrow="1" pageId="3" pageNumber="4">ITS1-OutF</th>
|
||
<td id="7692412EFF844B91D42FFAACA303FA96" box="[316,615,1318,1337]" gridcol="1" gridrow="1" pageId="3" pageNumber="4">TGCAATTATTGGTCGCGC</td>
|
||
<td id="7692412EFF844B91D7BBFAACA387FA96" box="[680,739,1318,1337]" gridcol="2" gridrow="1" pageId="3" pageNumber="4">54</td>
|
||
<td id="7692412EFF844B91D636FAACA296FA96" box="[805,1010,1318,1337]" gridcol="3" gridrow="1" pageId="3" pageNumber="4">Valcable</td>
|
||
<td id="7692412EFF844B91D126FAACA460FA96" box="[1077,1284,1318,1337]" gridcol="4" gridrow="1" pageId="3" pageNumber="4">
|
||
All
|
||
<taxonomicName id="4C735193FF84B46DD142FAACA5A5FA96" box="[1105,1217,1318,1337]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="genus">
|
||
<emphasis id="B907F602FF84B46DD142FAACA5A5FA96" box="[1105,1217,1318,1337]" italics="true" pageId="3" pageNumber="4">Trypanosoma</emphasis>
|
||
</taxonomicName>
|
||
mpeccem
|
||
</td>
|
||
<td id="7692412EFF844B91D055FAACA4F8FA96" box="[1350,1436,1318,1337]" gridcol="5" gridrow="1" pageId="3" pageNumber="4">
|
||
[
|
||
<bibRefCitation id="EFE257E1FF84B46DD05FFAACA404FA96" author="Mamcga DK & Hasem P & Blomcdge TJ & Gcbmon WC" box="[1356,1376,1318,1337]" pageId="3" pageNumber="4" pagination="909 - 18" refId="ref12333" refString="22. Mamcga DK, Smsth AJ, Hasem P, Blomcdge TJ, Gcbmon WC. Senmctcve detectcon of tlspanomomem cn tmetme flcem bs DNA amplcfccatcon. Int J Palamctol. 1992; 22: 909 - 18." type="journal article" year="1992">22</bibRefCitation>
|
||
]
|
||
</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584FAC1A4F8FAF1" box="[151,1436,1355,1374]" gridrow="2" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584FAC1A19DFAF1" box="[151,249,1355,1374]" gridcol="0" gridrow="2" pageId="3" pageNumber="4">ITS1-OutR</th>
|
||
<td id="7692412EFF844B91D42FFAC1A303FAF1" box="[316,615,1355,1374]" gridcol="1" gridrow="2" pageId="3" pageNumber="4">CTTTGCTGCGTTCTT</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584FAFBA4F8FA2B" box="[151,1436,1393,1412]" gridrow="3" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584FAFBA19DFA2B" box="[151,249,1393,1412]" gridcol="0" gridrow="3" pageId="3" pageNumber="4">ITS1-InF</th>
|
||
<td id="7692412EFF844B91D42FFAFBA303FA2B" box="[316,615,1393,1412]" gridcol="1" gridrow="3" pageId="3" pageNumber="4">TAGAGGAAGCAAAAG</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584FA1CA4F8FA06" box="[151,1436,1430,1449]" gridrow="4" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584FA1CA19DFA06" box="[151,249,1430,1449]" gridcol="0" gridrow="4" pageId="3" pageNumber="4">ITS1-InR</th>
|
||
<td id="7692412EFF844B91D42FFA1CA303FA06" box="[316,615,1430,1449]" gridcol="1" gridrow="4" pageId="3" pageNumber="4">AAGCCAAGTCATCCATCG</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584FA31A4F8FA61" box="[151,1436,1467,1486]" gridrow="5" pageId="3" pageNumber="4">
|
||
<th id="7692412EFF844B91D584FA31A19DFA61" box="[151,249,1467,1486]" gridcol="0" gridrow="5" pageId="3" pageNumber="4">TCON-OutF</th>
|
||
<td id="7692412EFF844B91D42FFA31A303FA61" box="[316,615,1467,1486]" gridcol="1" gridrow="5" pageId="3" pageNumber="4">TGCAATTATTGGTCGCGC</td>
|
||
<td id="7692412EFF844B91D7BBFA31A387FA61" box="[680,739,1467,1486]" gridcol="2" gridrow="5" pageId="3" pageNumber="4">54</td>
|
||
<td id="7692412EFF844B91D636FA31A296FA61" box="[805,1010,1467,1486]" gridcol="3" gridrow="5" pageId="3" pageNumber="4">681 (kclcfc) ol 781 (folemt)</td>
|
||
<td id="7692412EFF844B91D126FA31A460FA61" box="[1077,1284,1467,1486]" gridcol="4" gridrow="5" pageId="3" pageNumber="4">
|
||
<taxonomicName id="4C735193FF84B46DD126FA31A5C0FA61" box="[1077,1188,1467,1486]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="species" species="congolense">
|
||
<emphasis id="B907F602FF84B46DD126FA31A5C0FA61" box="[1077,1188,1467,1486]" italics="true" pageId="3" pageNumber="4">T. congolense</emphasis>
|
||
</taxonomicName>
|
||
</td>
|
||
<td id="7692412EFF844B91D055FA31A4F8FA61" box="[1350,1436,1467,1486]" gridcol="5" gridrow="5" pageId="3" pageNumber="4">
|
||
[
|
||
<bibRefCitation id="EFE257E1FF84B46DD05FFA31A404FA61" author="Mamcga DK & Hasem P & Blomcdge TJ & Gcbmon WC" box="[1356,1376,1467,1486]" pageId="3" pageNumber="4" pagination="909 - 18" refId="ref12333" refString="22. Mamcga DK, Smsth AJ, Hasem P, Blomcdge TJ, Gcbmon WC. Senmctcve detectcon of tlspanomomem cn tmetme flcem bs DNA amplcfccatcon. Int J Palamctol. 1992; 22: 909 - 18." type="journal article" year="1992">22</bibRefCitation>
|
||
]
|
||
</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584FA6BA4F8FA5B" box="[151,1436,1505,1524]" gridrow="6" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584FA6BA19DFA5B" box="[151,249,1505,1524]" gridcol="0" gridrow="6" pageId="3" pageNumber="4">TCON-OutR</th>
|
||
<td id="7692412EFF844B91D42FFA6BA303FA5B" box="[316,615,1505,1524]" gridcol="1" gridrow="6" pageId="3" pageNumber="4">TGCAATTATTGGTCGCGC</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F98CA4F8F9B6" box="[151,1436,1542,1561]" gridrow="7" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584F98CA19DF9B6" box="[151,249,1542,1561]" gridcol="0" gridrow="7" pageId="3" pageNumber="4">TCON-InF</th>
|
||
<td id="7692412EFF844B91D42FF98CA303F9B6" box="[316,615,1542,1561]" gridcol="1" gridrow="7" pageId="3" pageNumber="4">TCGCGTGTCTCACGT</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F9A1A4F8F991" box="[151,1436,1579,1598]" gridrow="8" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584F9A1A19DF991" box="[151,249,1579,1598]" gridcol="0" gridrow="8" pageId="3" pageNumber="4">TCON-InR</th>
|
||
<td id="7692412EFF844B91D42FF9A1A303F991" box="[316,615,1579,1598]" gridcol="1" gridrow="8" pageId="3" pageNumber="4">TCAAAGATTGGGCAATGT</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F9DBA4F8F9CB" box="[151,1436,1617,1636]" gridrow="9" pageId="3" pageNumber="4">
|
||
<th id="7692412EFF844B91D584F9DBA19DF9CB" box="[151,249,1617,1636]" gridcol="0" gridrow="9" pageId="3" pageNumber="4">TGR-OutF</th>
|
||
<td id="7692412EFF844B91D42FF9DBA303F9CB" box="[316,615,1617,1636]" gridcol="1" gridrow="9" pageId="3" pageNumber="4">TGGCAGACACATACCTGCCA</td>
|
||
<td id="7692412EFF844B91D7BBF9DBA387F9CB" box="[680,739,1617,1636]" gridcol="2" gridrow="9" pageId="3" pageNumber="4">54</td>
|
||
<td id="7692412EFF844B91D636F9DBA296F9CB" box="[805,1010,1617,1636]" gridcol="3" gridrow="9" pageId="3" pageNumber="4">526</td>
|
||
<td id="7692412EFF844B91D126F9DBA460F9CB" box="[1077,1284,1617,1636]" gridcol="4" gridrow="9" pageId="3" pageNumber="4">
|
||
<taxonomicName id="4C735193FF84B46DD126F9DBA515F9CB" box="[1077,1137,1617,1636]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="species" species="grayi">
|
||
<emphasis id="B907F602FF84B46DD126F9DBA515F9CB" box="[1077,1137,1617,1636]" italics="true" pageId="3" pageNumber="4">T. grayi</emphasis>
|
||
</taxonomicName>
|
||
</td>
|
||
<td id="7692412EFF844B91D055F9DBA4F8F9CB" box="[1350,1436,1617,1636]" gridcol="5" gridrow="9" pageId="3" pageNumber="4">Thcm mtuds</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F9FCA4F8F926" box="[151,1436,1654,1673]" gridrow="10" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584F9FCA19DF926" box="[151,249,1654,1673]" gridcol="0" gridrow="10" pageId="3" pageNumber="4">TGR-OutR</th>
|
||
<td id="7692412EFF844B91D42FF9FCA303F926" box="[316,615,1654,1673]" gridcol="1" gridrow="10" pageId="3" pageNumber="4">TGGGGATTACGGATGAAAC</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F911A4F8F901" box="[151,1436,1691,1710]" gridrow="11" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584F911A19DF901" box="[151,249,1691,1710]" gridcol="0" gridrow="11" pageId="3" pageNumber="4">TGR-InF</th>
|
||
<td id="7692412EFF844B91D42FF911A303F901" box="[316,615,1691,1710]" gridcol="1" gridrow="11" pageId="3" pageNumber="4">TTAAGGAGGCGCTCAGGTTC</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F94BA4F8F97B" box="[151,1436,1729,1748]" gridrow="12" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584F94BA19DF97B" box="[151,249,1729,1748]" gridcol="0" gridrow="12" pageId="3" pageNumber="4">TGR-InR</th>
|
||
<td id="7692412EFF844B91D42FF94BA303F97B" box="[316,615,1729,1748]" gridcol="1" gridrow="12" pageId="3" pageNumber="4">TGTGCATATACGTCTATG</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F96CA4F8F956" box="[151,1436,1766,1785]" gridrow="13" pageId="3" pageNumber="4">
|
||
<th id="7692412EFF844B91D584F96CA19DF956" box="[151,249,1766,1785]" gridcol="0" gridrow="13" pageId="3" pageNumber="4">TVIV-F</th>
|
||
<td id="7692412EFF844B91D42FF96CA303F956" box="[316,615,1766,1785]" gridcol="1" gridrow="13" pageId="3" pageNumber="4">CTGAGTGCTCCATGTCCCAC</td>
|
||
<td id="7692412EFF844B91D7BBF96CA387F956" box="[680,739,1766,1785]" gridcol="2" gridrow="13" pageId="3" pageNumber="4">60</td>
|
||
<td id="7692412EFF844B91D636F96CA296F956" box="[805,1010,1766,1785]" gridcol="3" gridrow="13" pageId="3" pageNumber="4">142</td>
|
||
<td id="7692412EFF844B91D126F96CA460F956" box="[1077,1284,1766,1785]" gridcol="4" gridrow="13" pageId="3" pageNumber="4">
|
||
<taxonomicName id="4C735193FF84B46DD126F96CA516F956" box="[1077,1138,1766,1785]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="species" species="vivax">
|
||
<emphasis id="B907F602FF84B46DD126F96CA516F956" box="[1077,1138,1766,1785]" italics="true" pageId="3" pageNumber="4">T. vivax</emphasis>
|
||
</taxonomicName>
|
||
</td>
|
||
<td id="7692412EFF844B91D055F96CA4F8F956" box="[1350,1436,1766,1785]" gridcol="5" gridrow="13" pageId="3" pageNumber="4">
|
||
[
|
||
<bibRefCitation id="EFE257E1FF84B46DD05FF96CA404F956" author="Adamm ER & Malele II & Mmangc AR & Gcbmon WC" box="[1356,1376,1766,1785]" pageId="3" pageNumber="4" pagination="103 - 9" refId="ref12412" refString="24. Adamm ER, Malele II, Mmangc AR, Gcbmon WC. Tlspanomome cdentcfccatcon cn wcld tmetme populatconm cn Tanzanca umcng genelcc plcmelm to amplcfs the lcbomomal RNA ITS 1 legcon. Acta Tlop. 2006; 100: 103 - 9." type="journal article" year="2006">24</bibRefCitation>
|
||
]
|
||
</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F881A4F8F8B1" box="[151,1436,1803,1822]" gridrow="14" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584F881A19DF8B1" box="[151,249,1803,1822]" gridcol="0" gridrow="14" pageId="3" pageNumber="4">TVIV-R</th>
|
||
<td id="7692412EFF844B91D42FF881A303F8B1" box="[316,615,1803,1822]" gridcol="1" gridrow="14" pageId="3" pageNumber="4">CCACCAGAACACCAACCTGA</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F8BBA4F8F8EB" box="[151,1436,1841,1860]" gridrow="15" pageId="3" pageNumber="4">
|
||
<th id="7692412EFF844B91D584F8BBA19DF8EB" box="[151,249,1841,1860]" gridcol="0" gridrow="15" pageId="3" pageNumber="4">COI-F</th>
|
||
<td id="7692412EFF844B91D42FF8BBA303F8EB" box="[316,615,1841,1860]" gridcol="1" gridrow="15" pageId="3" pageNumber="4">TTGATTTTTTGGTCATCCAGAAGT</td>
|
||
<td id="7692412EFF844B91D7BBF8BBA387F8EB" box="[680,739,1841,1860]" gridcol="2" gridrow="15" pageId="3" pageNumber="4">55</td>
|
||
<td id="7692412EFF844B91D636F8BBA296F8EB" box="[805,1010,1841,1860]" gridcol="3" gridrow="15" pageId="3" pageNumber="4">900</td>
|
||
<td id="7692412EFF844B91D126F8BBA460F8EB" box="[1077,1284,1841,1860]" gridcol="4" gridrow="15" pageId="3" pageNumber="4">
|
||
Genelcc
|
||
<taxonomicName id="4C735193FF84B46DD16FF8BBA586F8EB" box="[1148,1250,1841,1860]" class="Insecta" family="Glossinidae" genus="Glossina" kingdom="Animalia" order="Diptera" pageId="3" pageNumber="4" phylum="Arthropoda" rank="species" species="cox">
|
||
<emphasis id="B907F602FF84B46DD16FF8BBA586F8EB" box="[1148,1250,1841,1860]" italics="true" pageId="3" pageNumber="4">Glossina cox</emphasis>
|
||
</taxonomicName>
|
||
1
|
||
</td>
|
||
<td id="7692412EFF844B91D055F8BBA4F8F8EB" box="[1350,1436,1841,1860]" gridcol="5" gridrow="15" pageId="3" pageNumber="4">
|
||
[
|
||
<bibRefCitation id="EFE257E1FF84B46DD05FF8BBA404F8EB" author="Dsel NA & Lawton SP & Ravel S & Choc KS & Lehane MJ & Robcnmon AS" box="[1356,1376,1841,1860]" pageId="3" pageNumber="4" pagination="227 - 39" refId="ref12585" refString="28. Dsel NA, Lawton SP, Ravel S, Choc KS, Lehane MJ, Robcnmon AS, et al. Moleculal phslogenetccm of tmetme flcem (Dcptela: Glommcncdae) bamed on mctochondlcal (COI, 16 S, ND 2) and nucleal lcbomomal DNA mequencem, wcth an emphamcm on the palpalis gloup. Mol Phslogenet Evol. 2008; 49: 227 - 39." type="journal article" year="2008">28</bibRefCitation>
|
||
]
|
||
</td>
|
||
</tr>
|
||
<tr id="35432852FF844B91D584F8DCA4F8F8C6" box="[151,1436,1878,1897]" gridrow="16" pageId="3" pageNumber="4" rowspan-2="1" rowspan-3="1" rowspan-4="1" rowspan-5="1">
|
||
<th id="7692412EFF844B91D584F8DCA19DF8C6" box="[151,249,1878,1897]" gridcol="0" gridrow="16" pageId="3" pageNumber="4">COI-R</th>
|
||
<td id="7692412EFF844B91D42FF8DCA303F8C6" box="[316,615,1878,1897]" gridcol="1" gridrow="16" pageId="3" pageNumber="4">TGAAGCTTAAATTCATTGCACTAATC</td>
|
||
</tr>
|
||
</table>
|
||
</paragraph>
|
||
<tableNote id="76952B9EFF84B46DD584F8F6A022F80C" pageId="3" pageNumber="4" targetBox="[151,1436,1283,1897]" targetPageId="3">
|
||
<paragraph id="8BCC2A10FF84B46DD584F8F6A441F823" blockId="3.[151,1317,1915,1955]" box="[151,1317,1915,1932]" pageId="3" pageNumber="4">
|
||
<emphasis id="B907F602FF84B46DD584F8F6A199F824" box="[151,253,1916,1931]" italics="true" pageId="3" pageNumber="4">Abbreviations</emphasis>
|
||
: TCON,
|
||
<taxonomicName id="4C735193FF84B46DD42FF8F7A0FBF824" box="[316,415,1916,1931]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="species" species="congolense">
|
||
<emphasis id="B907F602FF84B46DD42FF8F7A0FBF824" box="[316,415,1916,1931]" italics="true" pageId="3" pageNumber="4">T. congolense</emphasis>
|
||
</taxonomicName>
|
||
; TGR,
|
||
<taxonomicName id="4C735193FF84B46DD4DDF8F7A367F824" box="[462,515,1916,1931]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="species" species="grayi">
|
||
<emphasis id="B907F602FF84B46DD4DDF8F7A367F824" box="[462,515,1916,1931]" italics="true" pageId="3" pageNumber="4">T. grayi</emphasis>
|
||
</taxonomicName>
|
||
; TVIV,
|
||
<taxonomicName id="4C735193FF84B46DD726F8F7A308F824" box="[565,620,1916,1931]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="3" pageNumber="4" phylum="Euglenozoa" rank="species" species="vivax">
|
||
<emphasis id="B907F602FF84B46DD726F8F7A308F824" box="[565,620,1916,1931]" italics="true" pageId="3" pageNumber="4">T. vivax</emphasis>
|
||
</taxonomicName>
|
||
;
|
||
<taxonomicName id="4C735193FF84B46DD767F8F7A3E9F824" box="[628,653,1917,1931]" class="Insecta" family="Glossinidae" genus="Glossina" kingdom="Animalia" order="Diptera" pageId="3" pageNumber="4" phylum="Arthropoda" rank="species" species="cox">
|
||
<emphasis id="B907F602FF84B46DD767F8F7A3E9F824" box="[628,653,1917,1931]" italics="true" pageId="3" pageNumber="4">cox</emphasis>
|
||
</taxonomicName>
|
||
1, cytochrome
|
||
<emphasis id="B907F602FF84B46DD7ECF8F7A263F824" box="[767,775,1917,1931]" italics="true" pageId="3" pageNumber="4">c</emphasis>
|
||
oxidase 1; Out, outer primer; In, inner primer;F, forward; R, reverse;TA,
|
||
</paragraph>
|
||
<paragraph id="8BCC2A10FF84B46DD584F818A022F80C" blockId="3.[151,1317,1915,1955]" box="[151,326,1938,1955]" pageId="3" pageNumber="4">annealing temperature</paragraph>
|
||
</tableNote>
|
||
<paragraph id="8BCC2A10FF83B46AD5BFFDE7A359FC0A" blockId="4.[151,776,236,933]" pageId="4" pageNumber="5">
|
||
Regardcng speccfcc cdentcfccatcon, the annealcng temperature was 54 °C for the fcrst reactcon and was varced durcng the second reactcon based on the meltcng temperatures of the prcmer sets 60 °C for
|
||
<taxonomicName id="4C735193FF83B46AD7B8FD47A263FD4B" box="[683,775,717,740]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="4" pageNumber="5" phylum="Euglenozoa" rank="species" species="vivax">
|
||
<emphasis id="B907F602FF83B46AD7B8FD47A263FD4B" box="[683,775,717,740]" italics="true" pageId="4" pageNumber="5">T. vivax</emphasis>
|
||
</taxonomicName>
|
||
and 54 °C for all other
|
||
<taxonomicName id="4C735193FF83B46AD4B4FD67A35BFCAB" box="[423,575,749,772]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="4" pageNumber="5" phylum="Euglenozoa" rank="genus">
|
||
<emphasis id="B907F602FF83B46AD4B4FD67A35BFCAB" box="[423,575,749,772]" italics="true" pageId="4" pageNumber="5">Trypanosoma</emphasis>
|
||
</taxonomicName>
|
||
specces. To optcmcse the
|
||
<collectionCode id="ED62B2D5FF83B46AD41BFC87A05DFC8A" box="[264,313,781,805]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
wcth prcmers speccfcc for
|
||
<taxonomicName id="4C735193FF83B46AD765FC87A3B4FC8B" box="[630,720,781,804]" class="Kinetoplastea" family="Trypanosomatidae" genus="Trypanosoma" kingdom="Protozoa" order="Trypanosomatida" pageId="4" pageNumber="5" phylum="Euglenozoa" rank="species" species="grayi">
|
||
<emphasis id="B907F602FF83B46AD765FC87A3B4FC8B" box="[630,720,781,804]" italics="true" pageId="4" pageNumber="5">T. grayi</emphasis>
|
||
</taxonomicName>
|
||
, the MgCl
|
||
<subScript id="17F72855FF83B46AD5C6FCBDA1BBFCE8" attach="left" box="[213,223,823,839]" fontSize="7" pageId="4" pageNumber="5">2</subScript>
|
||
concentratcon of the Dream
|
||
<emphasis id="B907F602FF83B46AD72AFCA7A307FCEB" box="[569,611,813,836]" italics="true" pageId="4" pageNumber="5">Taq</emphasis>
|
||
Green buffer (Thermo Sccentcfcc) was cncreased by addcng 2 mM MgCl
|
||
<subScript id="17F72855FF83B46AD5C6FCFDA1BBFC28" attach="left" box="[213,223,887,903]" fontSize="7" pageId="4" pageNumber="5">2</subScript>
|
||
. Amplcfced products were resolved by electrophorescs on 1.5% or 2% agarose gels.
|
||
</paragraph>
|
||
<paragraph id="8BCC2A10FF83B46AD584FC64A04AF92A" blockId="4.[151,776,1006,1669]" pageId="4" pageNumber="5">
|
||
<emphasis id="B907F602FF83B46AD584FC64A3ABFBAB" bold="true" box="[151,719,1006,1028]" pageId="4" pageNumber="5">
|
||
Purification and subcloning of selected
|
||
<collectionCode id="ED62B2D5FF83B46AD753FC64A30EFBAB" box="[576,618,1006,1028]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
products
|
||
</emphasis>
|
||
Selected
|
||
<collectionCode id="ED62B2D5FF83B46AD5EFFB87A049FB8A" box="[252,301,1037,1061]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
products were carefully exccsed from the gel uscng a clean scalpel. DNA was purcfced uscng GeneJet Gel Extractcon Kct (Thermo Sccentcfcc), followcng the cnstructcons of the manufacturer. DNA concentratcons were determcned at a wavelength of
|
||
<quantity id="4C8B87F5FF83B46AD738FB07A31BFB0A" box="[555,639,1165,1189]" metricMagnitude="-7" metricUnit="m" metricValue="2.6" pageId="4" pageNumber="5" unit="nm" value="260.0">260 nm</quantity>
|
||
on a Nanodrop 1000 apparatus (Thermo Sccentcfcc). Purcfced
|
||
<collectionCode id="ED62B2D5FF83B46AD7C5FB27A263FB6A" box="[726,775,1197,1221]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
products were cloned cnto ecther the lcnearczed plasmcd vector
|
||
<collectionCode id="ED62B2D5FF83B46AD5F5FB67A073FAAA" box="[230,279,1261,1285]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
<emphasis id="B907F602FF83B46AD404FB66A047FAAB" bold="true" box="[279,291,1260,1284]" pageId="4" pageNumber="5">™</emphasis>
|
||
2.1-
|
||
<collectionCode id="ED62B2D5FF83B46AD446FB67A0FBFAAA" box="[341,415,1261,1285]" pageId="4" pageNumber="5">TOPO</collectionCode>
|
||
(Thermo Sccentcfcc) wcth scngle 3
|
||
<emphasis id="B907F602FF83B46AD5B7FA86A1CAFA8B" box="[164,174,1292,1316]" italics="true" pageId="4" pageNumber="5">′</emphasis>
|
||
- deoxythymcdcne (
|
||
<collectionCode id="ED62B2D5FF83B46AD49FFA87A0FAFA8A" box="[396,414,1293,1317]" name="Tavera, Department of Geology and Geophysics" pageId="4" pageNumber="5">T</collectionCode>
|
||
) overhangs or lcnearcsed pJET 1.2/blunt plasmcd uscng the CloneJET
|
||
<collectionCode id="ED62B2D5FF83B46AD72FFAA7A309FAEA" box="[572,621,1325,1349]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
(Thermo Sccentcfcc), accordcng to the manufacturer
|
||
<emphasis id="B907F602FF83B46AD771FAC6A302FACB" box="[610,614,1356,1380]" italics="true" pageId="4" pageNumber="5">’</emphasis>
|
||
s cnstructcons. Posctcve clones were cdentcfced by colony
|
||
<collectionCode id="ED62B2D5FF83B46AD765FAE7A3C3FA2A" box="[630,679,1389,1413]" pageId="4" pageNumber="5">PCR</collectionCode>
|
||
and selected scngle colonces were cultured cn LB plus ampcccllcn (100 μg/ml) wcth shakcng overncght at 37 °C. Bacterca were collected by centrcfugatcon (4500×
|
||
<emphasis id="B907F602FF83B46AD75AFA47A330FA4B" box="[585,596,1485,1508]" italics="true" pageId="4" pageNumber="5">g</emphasis>
|
||
, 15 mcn at 4 °C) and the plasmcd DNA was purcfced uscng the NucleoBond Xtra Mcdc Plus McdcPrep Kct (Macherey-Nagel, Düren,
|
||
<collectingCountry id="F3646A80FF83B46AD584F9A7A199F9EA" box="[151,253,1581,1605]" name="Germany" pageId="4" pageNumber="5">Germany</collectingCountry>
|
||
), or GeneJET Plasmcd McncPrep Kct (Thermo Fcscher Sccentcfcc), accordcng to the cnstructcons of the manufacturer.
|
||
</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |