DNA molecular identification of Polyrhachis dives medicinal materials / 药学学报

In the study, specific primers were designed based on the CO Ⅰ gene sequence of Polyrhachis dives. By optimizing the genomic DNA extraction method and amplification conditions, we established an efficient, specific, and accurate DNA molecular identification method for Polyrhachis dives. In this method, the length of the target fragment was 294-308 bp, and the other counterfeits had no target bands. In this paper, the specific identification method of the origin of Polyrhachis dives established can be used to identify the medicinal materials of Polyrhachis dives accurately.

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.

Rapid identification of Cordyceps and its products using DNA barcoding method / 中国中药杂志

Cordyceps is one of the most valuable traditional Chinese medicines. There are various counterfeits in markets because of high price and limited output. In this study,116 Cordyceps,146 hosts and 29 related products were collected and detected by using normal DNA barcoding technology and specific PCR method. The results indicated that Cordyceps and its adulterants could be distinguished from each other through DNA barcoding technology based on ITS and COⅠsequences. Two pairs specific primers ITSSF1/ITSSR1 and ITSSF2/ITSSR2 were developed to amplify 297 bp and 136 bp ITS regions of Cordyceps sinensis,respectively. It could be used to identify C. sinensis specifically and rapidly. Furthermore,specific primers ITSSF1/ITSSR1 and ITSSF2/ITSSR2 combined with ITS and COⅠsequences could differentiate powder Cordyceps from fermentation mycelia and could identify related products. Therefore,the method developed from this study could be applied to identify the powder of Cordyceps from fermentation mycelia and related products efficiently.

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.

Analysis of population genetic diversity of mosquitoes from Shandong Provincebased on mitochondrial DNA cytochrome oxidase subunit Ⅰgene fragment / 中国血吸虫病防治杂志

Objective To explore the characteristics of gene sequence of mtDNA-COⅠof Culex pipiens pallens from differ-ent geographical regions in Shandong Province and different resistant strains from the lab and five common mosquito species, and analyze the genetic diversity of these mosquitoes.Methods Adult mosquitoes were collected from Jinan,Jining,Qingdao cities and other places in Shandong Province.The sensitive,dichlorvos-resistant,pyrethroid-resistant and propoxur-resistant strains were reared in the lab.Five species of mosquito(Cx.pipiens pallens,Cx.tritaeniorhynchus,Anopheles sinensis,Aedes al-bopictus,and Armigeres subalbatus)were collected from Jining City and identified in the lab.mtDNA-COⅠwas specifically am-plified by PCR and sequenced.The gene sequences were compared and analyzed by the biological information systems,and the phylogenetic tree was constructed.Results The amplified mtDNA-COⅠfragments of Cx.pipiens pallens from eight different cit-ies and four different resistant strains were 528 bp in length,with 67.4% A+T contents and two mutation sites.The nucleotide se-quence homology among the different geographic strains was 99.95% and the gene sequences of the four resistant strains were the same,showing a high homogeny.The amplified mtDNA-COⅠfragments of the five species of mosquitoes were 528 bp with 408 conserved sites,120 variable sites,42 parsimony informative sites and 78 singleton sites. The A+T contents were between 65.7% and 68.0%.The nucleotide sequence homology among the different mosquito species was between 86.17% and 92.05%,and the molecular identification was consistent with the traditional morphological identification. The molecular phylogenetic study showed that the different species were clustered at their own branch at the species and genus levels,while genera Armiger-es was distantly related to the others.Conclusion mtDNA-COⅠcould not serve as the molecular marker to analyze the popula-tion genetic variation and phylogenesis of Cx.pipiens pallens from different geographical regions and different resistant strains, but it has species and genus specificities,which could be used for the identification of the mosquito species and genus.

Identification of Fel Serpentis and its adulterants with specific PCR method / 中国中药杂志

The study aims at developing a convenient and specific method for the identification of Fel Serpentis DNA. The methods of Fel Serpentis genomic DNA purification were tested and optimized, four pairs of specific primers for the amplification of COⅠ, Cyt b and 16S were designed. Then the best pair of primers were selected according to the specificity and efficiency. The DNA fragment about 400 bp was amplified from 20 kinds of Fel Serpentis, whereas no DNA fragment was amplified from other animal samples under the same condition. This method is specific,accurate and reproducible, which provides a useful tool for the quality control of Fel Serpentis.

Multiplex PCR to simultaneous identification of five traditional Chinese medicinal seahorses / 中国中药杂志

Seahorse is one the most commonly used medicinal animal in China. Five species of Hippocampus are recorded as seahorse in the Chinese Pharmacopoeia. Because of the rapid decrease, several other Hippocampus species are often adulterants as medicinal seahorse in the herbal market, which compromise clinical efficacy and pose threat to endangered seahorse species conversation. Herein, a multiplex polymerase chain reaction (mPCR) method was developed to identify the biological sources of medicinal seahorses.Based on the sequences of mitochondrial DNA, five specific primers for Hippocampus trimaculatus, H. kelloggi, H. kuda, H. histrix and H. mohnikei (H. japonicus)were designed, respectively. Multiplex PCR yields the products of 155, 222, 292, 352, 458 bp amplicons in the present of DNA templates of H. kuda, H. mohnikei, H. kelloggi, H. histrix and H. trimaculatus, respectively. This multiplex PCR method which electrophoresis migration of different lengths of DNA bands allowed simultaneous identification of all the five medicinal seahorses in a single assay. It showed that this multiplex PCR assay is useful for the simultaneous identification the biological sources of complex multi-source samples, which could provide a useful tool for the quality control of seahorses.

Identification of sarcosaphagous flies by amplification and sequence analysis of COⅠ gene fragment from DNA extracts / 中国法医学杂志

Objective To identify the species of sarcosaphagous flies by amplifying cytochrome oxidase subunitI (CO I) gene fragment,combined with morphological characteristics.Methods The DNA of sarcosaphagous flies was extacted by modified Tris balanced phenol-Tris saturated phenol protocol,the amplificationand sequencing of CO Ⅰ fragment were conducted,and the results were then compared with the database for analysis.Results The modified DNA extration method by Tris balanced phenol-Tris saturated phenol could obtain effective DNA of sarcosaphagous flies,and be applied for CO Ⅰ fragment amplification,and thus for identification of the species of sarcosaphagous flies.Conclusion The DNA extracted from sarcosaphagous flies by modified Tris balanced phenol-Tris saturated phenol protocol could be used astemplate for the amplification of CO Ⅰ fragment,and the system could identify the species of sareosaphagous flies after sequencing and alignment with the sequences of database.Compared with traditional identification methods of using morphological characteristics,the current system is more accurate and could be more widely applied.

DNA molecular identification of Manis pentadactyla / 中国中药杂志

The study was aimed to establish a stable, accurate site specific PCR identification system to identify Manis pentadactyla and its adulterants using DNA molecular identification. The genomic DNA was extracted from experimental samples using the DNA extraction kit. The Cytb and CO Ⅰ genes were amplified using PCR and sequenced bi-directionally. Obtained sequences were assembled using the BioEdit software. The neighbor-joining tree was constructed by MEGA 6.0. Specific identification primers were designed according to the specific allelets, and PCR reaction system was optimized. The results indicated that the Cytb and CO Ⅰ sequence both were able to be used to identify M. pentadactyla and its adulterants. With the specific primers CO Ⅰ-S10/A5, the M. pentadactyla could be amplified a 400 bp DNA band when the annealing temperature ranged from 55 to 60 ℃ and the amount of DNA template ranged from 3 to 100 ng within 35 PCR cycles. However, other adulterants displayed no relevant bands. So that primers CO Ⅰ- S10 / A5 can be used to identify the M. pentadactyla with the adulterants.

Application of COⅠ Gene Mini-barcoding in Species Identification of Hair / 法医学杂志

OBJECTIVES@#To identify the species of mammalian hair using COⅠ gene mini-barcoding technology.@*METHODS@#A pair of universal primers for mammalian COⅠ gene mini-barcoding were designed. The hair DNA samples of experimental animals from 11 species in 5 orders, mammalia was amplified by PCR technology with universal primers, and the PCR products were sequenced by bi-directional DNA and after the sequences splicing the results were deposited into the BOLD database to perform the homology comparison.@*RESULTS@#The DNA of hair from all experiment animal species could be totally amplified by the mini-barcoding universal primers designed in this study. The length of amplified fragment was 147 bp. The result of homology comparison in the database showed that the closest matching species were consistent with the experiment animal species. In addition to the matching degree of Panthera leo （98.99%）, all homology matching degree of the other experiment animals were 100%, and the intraspecific genetic distance of Panthera leo was 1%. The interspecific genetic distance was ten times more than the intraspecific genetic distance which could be used for species identification.@*CONCLUSIONS@#The COⅠ gene mini-barcode technology is established and can provide fast and accurate species identification for hair of mammals.

Genetic Variation of Anopheles dirus A and D (Diptera:Culicidae) in China: Inferred by mtDNA-COⅠ Gene Sequences / 中国寄生虫学与寄生虫病杂志

Objective To interpret genetic variation and population structure of Anopheles dirus A and D from China by molecular marker. Methods Samples included An. dirus A of Hainan laboratory colony (n=13), and field specimen from Mengla (n=17) and Jiangcheng (n=17) in Yunnan Province. The specimens were identified by PCR assay before study. mtDNA-COⅠ region was amplified and sequenced. Genetic variation and population structure was estimated according to sequence data. Results The mtDNA-COⅠ gene with a length of 959 bp was analyzed. There were three haplotypes in An. dirus A and six haplotypes in An. dirus D. The above haplotypes distributed in three populations unif-ormly. The average number of pairwise differences within Mengla population (7.441 2) was greater than that of Jiangcheng (1.279 4) and Hainan (1.051 3) populations, which suggested that the level of genetic divergence was the highest within Mengla population. The result of hierarchical AMOVA estimation showed a limited geneflow (Fst=0.799 9), therefore the variation level in a population (20.01%) was smaller than among the populations (79.99%). Conclusion The inter-specific genetic variation between An. dirus A and D in China was small and the level of divergence among individuals was high.