Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae)
[Display omitted] ► Improving Demodex mite gDNA extraction and PCR examination technique. ► gDNA of single Demodex mite has been extracted successfully for the first time. ► It could satisfy the demands of Demodex 18S rDNA study at gene level. ► 18S rDNA is unsuitable for molecular identification at...
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| Veröffentlicht in: | Experimental parasitology 2012-05, Vol.131 (1), p.45-51 |
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| creator | Zhao, Ya-E. Xu, Ji-Ru Hu, Li Wu, Li-Ping Wang, Zheng-Hang |
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► Improving Demodex mite gDNA extraction and PCR examination technique. ► gDNA of single Demodex mite has been extracted successfully for the first time. ► It could satisfy the demands of Demodex 18S rDNA study at gene level. ► 18S rDNA is unsuitable for molecular identification at Demodex genus or below. ► 18S rDNA molecular data is suitable for family taxomomy in Cheyletoidea.
The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples (⩾1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs. |
| doi_str_mv | 10.1016/j.exppara.2012.02.025 |
| format | Article |
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► Improving Demodex mite gDNA extraction and PCR examination technique. ► gDNA of single Demodex mite has been extracted successfully for the first time. ► It could satisfy the demands of Demodex 18S rDNA study at gene level. ► 18S rDNA is unsuitable for molecular identification at Demodex genus or below. ► 18S rDNA molecular data is suitable for family taxomomy in Cheyletoidea.
The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples (⩾1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.</description><identifier>ISSN: 0014-4894</identifier><identifier>EISSN: 1090-2449</identifier><identifier>DOI: 10.1016/j.exppara.2012.02.025</identifier><identifier>PMID: 22414329</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>18S rDNA complete sequence analysis ; Animals ; Base Sequence ; Cloning, Molecular ; Demodex brevis ; Demodex canis ; Demodex folliculorum ; Demodex mite ; DNA, Ribosomal - chemistry ; DNA, Ribosomal - isolation & purification ; DNA-directed DNA polymerase ; dogs ; genetic distance ; Genome - genetics ; Genomic DNA extraction ; mites ; Mites - classification ; Mites - genetics ; Molecular Sequence Data ; parasitism ; parasitology ; PCR determination ; Phylogenetic relationship ; Phylogeny ; Polymerase Chain Reaction ; ribosomal DNA ; RNA, Ribosomal, 18S - genetics ; Sequence Alignment ; sequence analysis ; Sequence Analysis, DNA</subject><ispartof>Experimental parasitology, 2012-05, Vol.131 (1), p.45-51</ispartof><rights>2012 Elsevier Inc.</rights><rights>Copyright © 2012 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c389t-e672998bbd3c09a8374d61f47cb97165246a1ee38bbbfa08b1ed6754345c83b63</citedby><cites>FETCH-LOGICAL-c389t-e672998bbd3c09a8374d61f47cb97165246a1ee38bbbfa08b1ed6754345c83b63</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.exppara.2012.02.025$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22414329$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zhao, Ya-E.</creatorcontrib><creatorcontrib>Xu, Ji-Ru</creatorcontrib><creatorcontrib>Hu, Li</creatorcontrib><creatorcontrib>Wu, Li-Ping</creatorcontrib><creatorcontrib>Wang, Zheng-Hang</creatorcontrib><title>Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae)</title><title>Experimental parasitology</title><addtitle>Exp Parasitol</addtitle><description>[Display omitted]
► Improving Demodex mite gDNA extraction and PCR examination technique. ► gDNA of single Demodex mite has been extracted successfully for the first time. ► It could satisfy the demands of Demodex 18S rDNA study at gene level. ► 18S rDNA is unsuitable for molecular identification at Demodex genus or below. ► 18S rDNA molecular data is suitable for family taxomomy in Cheyletoidea.
The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples (⩾1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.</description><subject>18S rDNA complete sequence analysis</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Cloning, Molecular</subject><subject>Demodex brevis</subject><subject>Demodex canis</subject><subject>Demodex folliculorum</subject><subject>Demodex mite</subject><subject>DNA, Ribosomal - chemistry</subject><subject>DNA, Ribosomal - isolation & purification</subject><subject>DNA-directed DNA polymerase</subject><subject>dogs</subject><subject>genetic distance</subject><subject>Genome - genetics</subject><subject>Genomic DNA extraction</subject><subject>mites</subject><subject>Mites - classification</subject><subject>Mites - genetics</subject><subject>Molecular Sequence Data</subject><subject>parasitism</subject><subject>parasitology</subject><subject>PCR determination</subject><subject>Phylogenetic relationship</subject><subject>Phylogeny</subject><subject>Polymerase Chain Reaction</subject><subject>ribosomal DNA</subject><subject>RNA, Ribosomal, 18S - genetics</subject><subject>Sequence Alignment</subject><subject>sequence analysis</subject><subject>Sequence Analysis, DNA</subject><issn>0014-4894</issn><issn>1090-2449</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAQhi0EotvCTwB8bA_Z-itOzAWtti1UquBQerYce1J5lcTBzla7_x6HLFyRRrL06pmx_QxCHyhZU0Ll9W4Nh3E00awZoWxN5ipfoRUlihRMCPUarQihohC1EmfoPKUdIaSmTLxFZ4wJKjhTK3Tchn7sYAKc4NceBgvYDKY7Jp9waDGtH3G8-b7BjUngcBjwMwyh9xbPIRymaOzkc9zG0GM_OP_i3d50-Ab64OCAez9Bwpcba6L_vKTeemfg6h1605ouwfvTeYGe7m5_br8VDz--3m83D4XltZoKkBVTqm4axy1RpuaVcJK2orKNqqgsmZCGAvBMNK0hdUPByaoUXJS25o3kF-hymTvGkH-YJt37ZKHrzABhn3S2SYQiVPGMlgtqY0gpQqvH6HsTjxmaOal3-mRdz9Y1mavMfR9PV-ybHty_rr-aM_BpAVoTtHmOPumnxzyhzBuquPxDfFkIyCpePESdrJ_X4XwEO2kX_H8e8RuyU56O</recordid><startdate>20120501</startdate><enddate>20120501</enddate><creator>Zhao, Ya-E.</creator><creator>Xu, Ji-Ru</creator><creator>Hu, Li</creator><creator>Wu, Li-Ping</creator><creator>Wang, Zheng-Hang</creator><general>Elsevier Inc</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20120501</creationdate><title>Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae)</title><author>Zhao, Ya-E. ; Xu, Ji-Ru ; Hu, Li ; Wu, Li-Ping ; Wang, Zheng-Hang</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c389t-e672998bbd3c09a8374d61f47cb97165246a1ee38bbbfa08b1ed6754345c83b63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>18S rDNA complete sequence analysis</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Cloning, Molecular</topic><topic>Demodex brevis</topic><topic>Demodex canis</topic><topic>Demodex folliculorum</topic><topic>Demodex mite</topic><topic>DNA, Ribosomal - chemistry</topic><topic>DNA, Ribosomal - isolation & purification</topic><topic>DNA-directed DNA polymerase</topic><topic>dogs</topic><topic>genetic distance</topic><topic>Genome - genetics</topic><topic>Genomic DNA extraction</topic><topic>mites</topic><topic>Mites - classification</topic><topic>Mites - genetics</topic><topic>Molecular Sequence Data</topic><topic>parasitism</topic><topic>parasitology</topic><topic>PCR determination</topic><topic>Phylogenetic relationship</topic><topic>Phylogeny</topic><topic>Polymerase Chain Reaction</topic><topic>ribosomal DNA</topic><topic>RNA, Ribosomal, 18S - genetics</topic><topic>Sequence Alignment</topic><topic>sequence analysis</topic><topic>Sequence Analysis, DNA</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zhao, Ya-E.</creatorcontrib><creatorcontrib>Xu, Ji-Ru</creatorcontrib><creatorcontrib>Hu, Li</creatorcontrib><creatorcontrib>Wu, Li-Ping</creatorcontrib><creatorcontrib>Wang, Zheng-Hang</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Experimental parasitology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zhao, Ya-E.</au><au>Xu, Ji-Ru</au><au>Hu, Li</au><au>Wu, Li-Ping</au><au>Wang, Zheng-Hang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae)</atitle><jtitle>Experimental parasitology</jtitle><addtitle>Exp Parasitol</addtitle><date>2012-05-01</date><risdate>2012</risdate><volume>131</volume><issue>1</issue><spage>45</spage><epage>51</epage><pages>45-51</pages><issn>0014-4894</issn><eissn>1090-2449</eissn><abstract>[Display omitted]
► Improving Demodex mite gDNA extraction and PCR examination technique. ► gDNA of single Demodex mite has been extracted successfully for the first time. ► It could satisfy the demands of Demodex 18S rDNA study at gene level. ► 18S rDNA is unsuitable for molecular identification at Demodex genus or below. ► 18S rDNA molecular data is suitable for family taxomomy in Cheyletoidea.
The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples (⩾1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>22414329</pmid><doi>10.1016/j.exppara.2012.02.025</doi><tpages>7</tpages></addata></record> |
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| subjects | 18S rDNA complete sequence analysis Animals Base Sequence Cloning, Molecular Demodex brevis Demodex canis Demodex folliculorum Demodex mite DNA, Ribosomal - chemistry DNA, Ribosomal - isolation & purification DNA-directed DNA polymerase dogs genetic distance Genome - genetics Genomic DNA extraction mites Mites - classification Mites - genetics Molecular Sequence Data parasitism parasitology PCR determination Phylogenetic relationship Phylogeny Polymerase Chain Reaction ribosomal DNA RNA, Ribosomal, 18S - genetics Sequence Alignment sequence analysis Sequence Analysis, DNA |
| title | Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae) |
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