Identification of Common Sarcosaprophagous Flies in the Yangtze River Delta by COⅠ Gene / 法医学杂志

OBJECTIVES@#To identify the common sarcosaprophagous flies in the Yangtze River Delta based on mitochondrial cytochrome c oxidase subunit Ⅰ(COⅠ) gene sequence and verify the reliability of this method.@*METHODS@#Seven common genetically stable sarcosaprophagous flies in three families and six genera were collected from large domestic pig carcasses placed in the field and cultured in the laboratory for many generations. The whole genome DNA was extracted and the COⅠ gene fragment was amplified. The forward and reverse sequencing was followed by splicing. The base composition of the amplified fragment and the rate of interspecific evolutionary divergence were analyzed by software such as Mega 7.0.26. The phylogenetic tree of COⅠ gene sequence of common necrophagous flies in the Yangtze River Delta was established by neighbor joining (NJ) method and unweighted pair-group method with arithmetic means (UPGMA) method.@*RESULTS@#The average base composition of different flies was A(30.14%), T(38.23%), C(15.98%), G(15.65%). The rate of interspecific evolutionary divergence ranged from 2.2% to 15.3%, the lowest rate was between Chrysomya megacephala and Chrysomya pinguis, the highest rate was between Muscina stabulans and Boettcherisca peregrina.@*CONCLUSIONS@#COⅠ gene can be used to identify the common necrophagous flies in the Yangtze River Delta.

Identification of 24 Rana Species Including Rana dybowskii and Rana chensinensis Based on COⅠ Sequences / 中国实验方剂学杂志

Objective:To identify 24 <italic>Rana</italic> species such as <italic>Rana dybowskii</italic> by mitochondrial cytochrome C oxidase subunit I （<italic>CO</italic>Ⅰ） gene-based DNA barcoding and build the neighbour-joining （NJ） tree for hierarchical cluster analysis， so as to provide a basis for the identification and classification of <italic>Rana</italic> species as well as the discovery of new species. Method:<italic>R. dybowskii</italic>， <italic>R. chensinensis</italic>， <italic>R. amurensis</italic>， <italic>R. culaiensi</italic>s， and <italic>R. huanrenesis</italic>， ten for each species， were collected for DNA extraction and polymerase chain reaction （PCR） amplification<italic> </italic>and sequencing. A total of 50 <italic>CO</italic>Ⅰ gene sequences were obtained. Then 163 <italic>CO</italic>Ⅰ gene sequences for 24 species of <italic>Rana</italic> and one <italic>CO</italic>Ⅰ gene sequence for <italic>Pelophylax</italic>，<italic> Odorrana</italic>， <italic>Nidirana</italic>， <italic>Hylarana</italic>， and <italic>Amolops</italic> were harvested from GenBank. After sequence alignment by MEGA X， the parsimony-informative sites of <italic>CO</italic>Ⅰ gene sequences were analyzed and the intraspecific and interspecific genetic distances were calculated， followed by the built of NJ tree and hierarchical cluster analysis. Result:The <italic>CO</italic>Ⅰ gene sequences of 24<italic> Rana</italic> species including <italic>R. dybowskii</italic> were 554 bp in length and there were 210 parsimony-informative sites in total. The intraspecific genetic distance of each species was smaller than 2%. Except that the interspecific genetic distance between <italic>R. sangzhiensis</italic> and <italic>R. zhengi</italic> was 0.004， the genetic distances between the other species ranged from 0.024 to 0.228. <italic>R. sangzhiensis</italic> and <italic>R. zhengi</italic> were clustered into one branch and some <italic>R. dybowskii</italic> and <italic>R. uenoi</italic> into one branch. There were two separate branches for <italic>R. chensinensis</italic> and the other species were all clustered independently. Conclusion:<italic>CO</italic>Ⅰ-based DNA barcoding enabled the identification of 24 species of <italic>Rana</italic> including <italic>R.dybowskii</italic>. The findings supported that <italic>R. sangzhiensis</italic>， <italic>R. zhengi</italic>， <italic>R. coreana</italic>， and <italic>R. kunyuensis</italic> were the same species. One branch of <italic>R. chensinensis </italic>might be one of the four undownloaded species in Ranidae or a new species. The results have demonstrated that <italic>CO</italic>Ⅰ-based DNA barcoding allows not only the identification of 24 species of Rana including <italic>R. dybowskii </italic>but also the classification of ranidae species and the discovery of new species or subspecies.

COⅠ and 16S rDNA Sequence Identification of Common Necrophagous Flies in Fujian Province / 法医学杂志

Objective To identify the species of common necrophagous flies in Fujian Province by gene fragment sequences of mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） and 16S ribosomal deoxyribonucleic acid （16S rDNA）, and to explore the identification efficacy of these two molecular markers. Methods In total 22 common necrophagous flies were collected from the death scenes in 9 different regions in Fujian Province and DNA was extracted from the flies after morphological identification. The gene fragments of COⅠ and 16S rDNA were amplified and sequenced. All the sequences were uploaded to GeneBank and BLAST and MEGA 10.0 software were used to perform sequence alignment, homology analysis and intraspecific and interspecific genetic distance analysis. The phylogenetic trees of DNA fragment sequences of COⅠ and 16S rDNA of common necrophagous flies in Fujian Province were established by unweighted pair-group method with arithmetic means （UPGMA）, respectively. Results The flies were classified into 6 species, 5 genera and 3 families by morphological identification. The results of gene sequence analysis showed that the average number of interspecific and intraspecific genetic distance of 16S rDNA ranged from 1.8% to 8.9% and 0.0% to 2.4%, respectively. The average number of interspecific and intraspecific genetic distance of COⅠ ranged from 7.2% to 13.6% and 0.0% to 6.3%, respectively. Conclusion The gene sequences of COⅠ and 16S rDNA can accurately identify the species of different necrophagous flies, and 16S rDNA showed higher value in species identification of common calliphoridae necrophagous flies in Fujian Province.

Analysis of 28S rRNA and COⅠ Gene Sequence of Nine Necrophagous Calliphorid Flies from Luoyang / 法医学杂志

Objective To assess the feasibility of using 28S ribosomal RNA （28S rRNA） and mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） gene sequences of nine necrophagous Calliphorid flies for the identification of common necrophagous Calliphorid flies, and to provide technical support for postmortem interval （PMI） estimation. Methods Twenty-three Calliphorid flies were collected and identified morphologically, and DNA were extracted from legs. The gene fragments of 28S rRNA and COⅠ were amplified and sequenced, then the sequence alignment was performed with BLAST. The composition of obtained sequences was analyzed and evolutionary divergence rate between species and intraspecies were established. The phylogeny tree was constructed with neighbor-joining method. Results The 23 necrophagous Calliphorid flies were identified to 9 species of 5 genera. The 715 bp from 28S rRNA and 637 bp from COⅠ gene were obtained and the online BLAST result showed more than 99% of similarity. The phylogeny tree showed that the necrophagous flies could cluster well into 9 groups, which was consistent with morphological identification results. The intraspecific difference in 28S rRNA was 0 and the interspecific difference was 0.001-0.033. The intraspecific difference in COⅠ was 0-0.008 and the interspecific difference was 0.006-0.101. Conclusion Combined use of 28S rRNA and COⅠ gene sequence fragments can effectively identify the nine Calliphorid flies in this study. However, for closely related blowfly species, more genetic markers should be explored and used in combination in future.

Molecular identification of main vector fleas in Qinghai plague foci by DNA barcoding / 中华地方病学杂志

Objective To make up the limitations of traditional morphological classification methods,we identified vector fleas by DNA barcoding in Qinghai Province.Methods The mt DNA cytochrome c oxidase subunit Ⅰ (CO Ⅰ) gene was amplified by PCR from 36 muscle tissues of fleas in 3 states,2 cities and 5 counties of Qinghai Province,and the obtained CO Ⅰ gene fragments were sequenced and aligned.The intra-and inter-species genetic distances were calculated with Mega 6 software using K2-P model and a phylogenetic tree was constructed with neighbor-joining (NJ) method.Results Totally 36 CO Ⅰ gene sequences of 2 superfamilies,4 genera and 6 kinds of vector fleas were measured,the average genetic distance was 0.119,and the intraspecific distance was 0.002-0.027,the interspecific distance was 0.039-0.207,and the interspecific genetic distance was significantly greater than the intraspecific genetic distance.NJ tree showed the same species had formed a single line with high support rate and interspecific branch was clear.Conclusion DNA barcoding is suitable for identification of vector fleas in Qinghai Province,may make up the limitations of traditional morphological classification methods.

Study on DNA Barcode Identification Method of Saiga tatarica and Its Adulterants Based on CO Ⅰ Gene / 中国药房

OBJECTIVE:To investigate the feasibility of molecule identication of Saiga tatarica and its adulterants by using cytochrome C oxidase subunit Ⅰ (CO Ⅰ) gene.METHODS:A total of 7 horns and incomplete horns were collected from 4 areas.The extraction effect of DNA from bone plug and stratum corneum were investigated;PCR technology was used to amplify CO Ⅰ gene of samples using universal primer LCO Ⅰ 490,HCO2198;after gel electrophoresis,purification (750 bp strip) and sequencing,using CO Ⅰ gene as barcode alignment sequence,online comparison was conducted by using Blast software of NCBI database to determine specific species.RESULTS:The extraction of DNA from stratum corneum was better (DNA concentration was 15.7-22.6 ng/μL,the absorbance of 260 nm/280 nm was 1.73-4.72).Online comparison showed that the similarity of CO Ⅰ gene in all sampies reached 99％,mainly from the horns of saiga antelope,Tibetan antelope,gazelle,sheep and goats.CONCLUSIONS:DNA barcode technology based on CO Ⅰ gene can be used for the identification of S.tatarica and its adulterants.The technology can provide an accurate and objective method for the identification of horn medicinal materials.

Genotyping and polymorphism analysis of cytochrome c oxidase subunit Ⅰgene of Pomacea canaliculata from Lincang City in Yunnan Province / 中国血吸虫病防治杂志

Objective To analyze the genetic diversity of Pomacea canaliculata based on the mitochondria DNA cytochrome c oxidase subunitⅠ(mtDNA COⅠ)gene as a molecular marker in Lincang City of Yunnan Province,so as to provide the scien-tific data for monitoring Angiostrongylus cantonensis in local areas. Methods The genotypes and polymorphisms of 38 speci-mens of P.canaliculata collected from Mengding Town of Lincang City were analyzed by sequencing COⅠgene.The phylogenet-ic tree and genetic distances were produced based on the haplotypes from GenBank and the present study by using the neighbour-joining method with the software MEGA version 6.06. Results Totally 31 sequences were acquired in the present study and they produced 3 unique haplotypes.Haplotype 1 showed a higher frequency compared to the others and it accounted for 83.9 % (26/31).The data showed that the least genetic distances ranged from 0 to 0.052 between P.canaliculata and 3 haplotypes,as well as the largest genetic distances ranged from 0.021 to 0.239 between Pila conica and 3 haplotypes.Otherwise,the analysis of the phylogenetic trees based on COⅠgene sequences of P.canaliculata indicated that all of 3 haplotypes clustered into one big clade with that from Japan(GenBank accession number: AB433769),China(GenBank accession number: KT313034)and USA(GenBank accession number:EU523129),which owned the closet relationship amongst them.Their genetic relationships were distantly related to the GenBank's reference sequences of P.insularum(GenBank accession number:EF514942),P.cam-ena(GenBank accession number: EF515059)and so on. Conclusion There is a P. canaliculata species in Lincang City of Yunnan Province as well as a high genetic diversity amongst the acquired 3 haplotypes in this study.

Identification of rodent animals by DNA barcoding technology in Gansu Province / 中华地方病学杂志

Objective To apply the DNA barcoding technology for identification of rodent animals and to establish a rodent animal DNA barcode database in Gansu Province.Methods A total of 54 rodent animals were detected.DNA barcoding technology was used to analyze the DNA mitochondrial cytochrome C oxidase subunit Ⅰ (CO Ⅰ) gene sequence in Gansu Province.Results The intra-specific genetic distance was 0-2％ while the interspecific distance ranged from 18％ to 30％.Eight major clusters were apparently showed on a Neighbor joining tree.Conclusion DNA barcoding technology could overcome the shortcomings of the morphological identification,so it could be used to identify the rodent animals and has important implications for disease control and prevention in the natural focus of Gansu Province.

Genetic diversity of cytochrome C oxidase subunit Ⅰ genes in Aedes albopictus from dengue surveillance sites in Fujian Province, China / 中国人兽共患病学报

The mosquito Aedes albopictus is the primary vector for dengue virus transmission in Fujian Province.Despite that active dengue surveillance has been launched in several sites since 2005,the genetic diversity of A.albopictus from these sites remains exclusive.In this study,mosquito pools collected from dengue surveillance sites during 2015-2016 were randomly selected,the full-length mitochondrial cytochrome C oxidase subunit Ⅰ (mtDNA-COⅠ) were amplified and sequenced.Preliminary sequence alignment of 12 amplicons with the reference sequence indicated 99 ％ homology at nucleotide level,due to varia tions at 9 nucleotide sites.Three haplotypes,namely H02,H03 and H08,were determined based on phylogenetic analysis with 72 reference sequences of known haplotypes.These observations facilitate surveillance and control of arboviral diseases in Fujian.

Amplification of cytochrome C oxidase subunit Ⅰ gene of Brandt's Vole by nested PCR / 中华地方病学杂志

Objective To determine a method for amplification of cytochrome C oxidase subunit Ⅰ (CO Ⅰ) gene of Brandt's vole.Methods 发he Brandt's Voles were captured in Abagaqi Xilingol League Inner Mongolia,and DNA was extracted from liver tissue.CO Ⅰ gene was amplified by nested PCR and sequenced afterwards.Results A band of 657 bp and 1 132 bp was amplified by internal and external PCR primers,respectively,which were consistent with expected sizes.A total of 12 segments of Brandt's Vole CO Ⅰ gene sequences were amplified by PCR and verified by sequencing.一he sequence number was KF182196-KF182207 in GenBank.After gene sequence alignment of the 12 CO Ⅰ gene sequences,it was found that the similarity was 100％,and no base mutation.Conclusion CO Ⅰ gene of Brandt's Vole could be amplified by nested PCR without pseudo gene.

Application of DNA barcoding technique of COXⅠ gene in identification of ticks / 国际检验医学杂志

Objective To investigate the application of the cytochrome C oxidase subunit Ⅰ gene(COXⅠ ) barcoding in the i‐dentification of wild‐caught ticks .Methods The COXⅠ gene of 9 caught tick individuals with different species was sequenced ,the DNAstar software was used to conduct the sequence comparison and homology analysis .Meanwhile the philp3 .6 software was a‐dopted to select the maximal likelihood(ML) method for constructing the evolutionary trees .Results In the phylogenetic trees ,the samples of sh2011040702 ,ra2011042193 and ra2011051168 with Haemaphysalis were in a branch ,sh2011051404 ,sh2011051408 and ra2011041177 with dermacentor were clustered together ,and sh2011051837 ,sh2011041917 and ra2011041175 with hyalomma were in a branch ,which were consistent with the morphological identification results .Conclusion Making the COXⅠ gene sequence as tick DNA barcoding has certain feasibility for identifying the tick species .

Identification on host animals for plague by DNA barcoding technology in Shaanxi province / 中华流行病学杂志

Objective To apply the DNA barcoding technology for identification on host animal and to establish the host animal DNA bar code database on natural foci of plague in Shaanxi.Methods 139 host animals belonging to 3 orders,6 families and 12 genera and 62 residues belonging to 7 species from 8 different parts of the province,were detected.DNA barcoding technology was used to analyze the DNA CO Ⅰ gene sequence on the natural foci of plague in Dingbian county.Results The intra-specific genetic distance was less than 2％ while the inter-specific distance ranged from 8.9％ to 15.1％.Fourteen major clusters were apparently showed on a Neighbor-Joining tree.Residue samples could be detected regarding the objective gene.Alashan ground squirrel was previously noticed to carry 14 major clusters,which were previously mistakenly named as Citellus dauricus in Dingbian county.Conclusion DNA barcoding technology could overcome the shortcomings caused by the morphological identification so could be used to identify the host animal and residues in the natural focus of plague.

Cloning and sequence analysis of mtDNA cytochrome c oxidase subunitⅠ gene of the Asian Black Bear Sichuan Subspecies / 第三军医大学学报

Objective To explore the features of the cytochrome c oxidase subunit Ⅰ (COXⅠ) of Asian black bear Sichuan subspecies. Methods Primers were designed according published gene sequences of other bear species. A DNA sequence was amplified by PCR from the bear Sichuan subspecies’s hair-follicle tissue total DNA, then the PCR product was sequenced. The results were analyzed and a forecast analysis was carried on. Results The amplified COXⅠ gene of Sichuan black bear was 1 605 bp and contained a 1545-nucleotide open reading frame encoding 514 amino acid residues. The overall base composition of COXⅠ gene is 26.9 %, 23.1%, 18.3 %, and 31.8 % respectively to A, C, G, and T. The start codon of the COXⅠ is ATG, while the termination codon TAA. The protein’s pI is 6.29 and weight is 57.2?10 3 . The similarity analysis and phylogenetic tree analysis indicated that Sichuan black bear has closer relationship to American black bear, but not to brown bear and polar bear. Among dogs, caws and rats, it is closer to the former one. Conclusion There are high similarities in the DNA sequence, protein amino acid sequence of Asian black bear Sichuan subspecies with other bear species, indicating a high conservativity of COXⅠ in these involved species.