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 Characterization of Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae) from Goats in the Western Part of India by LSU of Nuclear Ribosomal DNA

The rumen parasite, Gastrothylax crumenifer (Platyhelminthes: Gastrothylacidae), is a highly pathogenic trematode parasite of goat (Capra hircus). It sucks blood that causes acute disease like anemia, and severe economic losses occur due to morbidity and mortality of the ruminant infected by these worms. The study of these rumen paramphistomes, their infection, and public health importance remains unclear in India especially in the western part of state Uttar Pradesh (U.P.), Meerut, India, where the goat meat consumption is very high. This paper provides the molecular characterization of G. crumenifer recovered from the rumen of Capra hircus from Meerut, U.P., India by the partial sequence of 28S rDNA. Nucleotide sequence similarity searching on BLAST of 28S rDNA from parasites showed the highest identity with those of G. crumenifer from the same host Capra hircus. This is the first report of molecular identification of G. crumenifer from this part of India.

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.

Identification of Clinical Mold Isolates by Sequence Analysis of the Internal Transcribed Spacer Region, Ribosomal Large-Subunit D1/D2, and beta-Tubulin

BACKGROUND: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. METHODS: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the beta-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. RESULTS: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and beta-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of beta-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. CONCLUSIONS: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.

Molecular Variation and Distribution of Anopheles fluviatilis (Diptera: Culicidae) Complex in Iran

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.

Identification of parasite DNA in common bile duct stones by PCR and DNA sequencing

We attempted to identify parasite DNA in the biliary stones of humans via PCR and DNA sequencing. Genomic DNA was isolated from each of 15 common bile duct (CBD) stones and 5 gallbladder (GB) stones. The patients who had the CBD stones suffered from cholangitis, and the patients with GB stones showed acute cholecystitis, respectively. The 28S and 18S rDNA genes were amplified successfully from 3 and/or 1 common bile duct stone samples, and then cloned and sequenced. The 28S and 18S rDNA sequences were highly conserved among isolates. Identity of the obtained 28S D1 rDNA with that of Clonorchis sinensis was higher than 97.6%, and identity of the 18S rDNA with that of other Ascarididae was 97.9%. Almost no intra-specific variations were detected in the 28S and 18S rDNA with the exception of a few nucleotide variations, i.e., substitution and deletion. These findings suggest that C. sinensis and Ascaris lumbricoides may be related with the biliary stone formation and development.

Molecular phylogeny of parasitic Platyhelminthes based on sequences of partial 28S rDNA D1 and mitochondrial cytochrome c oxidase subunit I

The phylogenic relationships existing among 14 parasitic Platyhelminthes in the Republic of Korea were investigated via the use of the partial 28S ribosomal DNA (rDNA) D1 region and the partial mitochondrial cytochrome c oxidase subunit 1 (mCOI) DNA sequences. The nucleotide sequences were analyzed by length, G + C %, nucleotide differences and gaps in order to determine the analyzed phylogenic relationships. The phylogenic patterns of the 28S rDNA D1 and mCOI regions were closely related within the same class and order as analyzed by the PAUP 4.0 program, with the exception of a few species. These findings indicate that the 28S rDNA gene sequence is more highly conserved than are the mCOI gene sequences. The 28S rDNA gene may prove useful in studies of the systematics and population genetic structures of parasitic Platyhelminthes.

Sequence analyses of three nuclear ribosomal loci and a mitochondrial locus in cytologically different forms of Thai Anopheles aconitus mosquitoes.

Ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of Anopheles aconitus mosquitoes were examined to investigate intra- and inter-species variation amongst the members of the Minimus group of Anopheles subgenus Cellia. Three rDNA loci (ITS1, ITS2 and D3 regions) and a mtDNA locus (cytochrome oxidase II) were analyzed in An. aconitus Form B and Form C collected in Chiang Mai Province, Thailand. The results show that the consensus sequences of the four loci of the two forms are consistent with those of mosquitoes in the genus Anopheles. No intraindividual variation was detected, but intrapopulation variation was present with polymorphic sequences in some forms for each gene examined. The variation rates were approximately 0.15 to 0.8%. These data indicate that An. aconitus Form B and Form C in Chiang Mai, Thailand are conspecific. In this study, the complete ITS1 sequence of An. aconitus is reported for the first time. The region showed a high variation rate (approximately 55%), compared to the closely related species An. minimus C. It is suggested that this rDNA locus may provide sequence information to differentiate the members of the Minimus group of Anopheles subgenus Cellia.

Molecular variation and phylogeny of the Anopheles minimus complex (Diptera: Culicidae) inhabiting Southeast Asian countries, based on ribosomal DNA internal transcribed spacers, ITS1 and 2, and the 28S D3 sequences.

Anopheles minimus (Theobald) is one of the most important vectors of human malaria in Southeast Asia. Morphological studies now have revealed five sibling species as its complex, designated as species A to E. The present study investigated the genetic divergence among An. minimus populations from four countries (Japan, China, Thailand and Indonesia), based on the DNA sequences data of the D3 (the third domain of the 28S ribosomal gene) and ITS2 (the second internal transcribed spacer of the ribosomal gene) is reported. The D3 and ITS2 phylogenetic trees, and the electrophoretic profile of ITS1 (the first internal transcribed spacer of the ribosomal gene) indicated that our An. minimus populations are comprised of three groups: the Japanese population as group I, the population from Guangxi Province of China (GX population) as group II, and others, as group III. The results showed the morphological similarity of group III and GX with the species complex A and B, respectively. It is interesting that both two species A (YN population) and species B (GX) occur in China, and that both species, An. minimus species A (LB-95 population) and the closer population An. flavirostris (Ludlow) (LB-00 population) appeared to be present on the Lombok Island of Indonesia, although in far separated localities. Moreover, this molecular evidence confirms the previous suggestion that the population from the Ishigaki Island of Japan should be classified as a new genetic status species E.

DNA-based technique and species identification / 法医学杂志

The species identification plays a key role in forensic analysis. Generally, three methods have been applied for this purpose, they are morphologic-based, serologic-based and DNA-based techniques. This review mainly discussed the DNA-based technique and evaluate it's value in species identification of forensic science.