Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

ABSTRACT Background: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. Methods: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. Results: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. Conclusions: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

Study on Identification Methods of Bullwhip by PCR Technology Based on Cyt b Gene / 中国药房

OBJECTIVE： To establish the method for PCR identification of bullwhip， and to identify the authenticity of bullwhip at the molecular level. METHODS： DNA samples of bullwhip and its counterfeits （donkey whip， pig whip， sheep whip） were extracted and their integrity， purity and concentration were detected. Using GenBank related information， using mitochondrial cytochrome b （Cyt b） gene of bullwhip as target gene， Primer-BLAST online software was used to design specific primer. PCR amplification was performed for whips of different species， and electrophoretic analysis was conducted for the product. PCR products of bullwhip samples were cloned and confirmed by DNA sequencing. The specificity and repeatability of the established PCR method were verified. RESULTS： DNA purity of the bullwhip and its counterfeits was high， and there was no protein or RNA pollution. 1.5％ agarose gel electrophoresis showed that there were obvious target gene bands of bullwhip samples at 200-300 bp， while no corresponding bands appeared in other counterfeit products. The results of DNA sequencing showed that the nucleotide sequence of the gene fragment of bullwhip was 100％ similar to that of the bullwhip in GeneBank. Results of methodological validation showed that established method was specific and reproducible. CONCLUSIONS： The established PCR identification method based on Cyt b gene in the study is simple， rapid， accurate， specific and reproducible， and can meet the requirements of analysis and identification of bullwhip and its counterfeits.

Cytochrome b Diversity and Phylogeny of Six Egyptian Sheep Breeds

Aim: Cytochrome b (Cyt-b) regions of mtDNA have received more attention due to their role in the genetic diversity and phylogenetic studies in different livestock. By using Cytochrome b sequencing, we aimed to clarify the genetic affinities and phylogeny of six Egyptian sheep breeds. Methodology: The genomic DNA was extracted and the specific primers were used for conventional PCR amplification of the Cytochrome b region of mtDNA (1134-bp) in sheep. The alignment of sequences was done to identify the sequence variations and polymorphic sites in the amplified fragments. Results: The results showed the presence of 39 polymorphic sites leading to the formation of 29 haplotypes (accession numbers: MG407500 - MG407528) with total haplotype diversity 0.814 and nucleotide diversity 0.00359. The lowest genetic distance was observed between Rahmani and Saidi while the highest distance was observed between Ossimi and Sohagi. The sequences of 111 analyzed samples were aligned with reference sequences of different haplogroups; A, B, C, D and E. The result showed that 86 out of 111 tested animals cluster with haplogroup B (77.48%), whereas 12 tested animals cluster with each of both haplogroups A and C (10.81%) and one animal belongs to haplogroup E (0.90%) with the absence of haplogroup D. Conclusion: Cytochrome b regions of mtDNA have an important role in the genetic diversity and phylogenetic studies in farm animals as well as many other mammalian species.

Application progress on mitochondrial DNA markers in identification of animal medicinal materials / 中草药

Animal medicinal materials are important parts of traditional Chinese medicine (TCM) with a long history and remarkable efficacy. Due to the destruction of wild animal resources and increasingly market demands, the adulterations of animals medicinal materials have become more common in medicine market and bring security risks for clinical application. In recent years, mitochondrial DNA is widely used in the field of animal population genetics, phylogeography, and phylogenetic development due to its maternally inherited features and abundant genetic diversity, and has achieved fruitful results in the field of molecular identification, which provides technical support for quality control of animal medicinal materials. This paper summarizes the application value and research status of COI, Cyt b, and 12S rRNA in the identification of animal medicinal materials, and gives a brief discussion on the follow-up development of mtDNA marker technique in the identification of animal medicinal materials in combination with previous result, which provides technical support for reasonable utilization of medicinal animal resources.

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.

Genetic characteristics of Spermophilus in Shaanxi Province, China / 中国人兽共患病学报

We studied the genetic characteristics of Spermophilus in Shaanxi Province,China.The COI,Cyt-b gene were sequenced and the results were compared with those of dauricus from Inner Mongolia Keyouzhong Banner and Zhengxiangbai Banner,and S.alaschanicus from Haiyuan County of Ningxia.And genetic distance was analyzed and Neighbor-Joining tree was built.Results showed that the genetic distance of COI gene sequences between Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia was ≤0.5％,and the genetic distance was ranged from 7.9％ to 9.3％ with Citellus dauricus from Inner Mongolia.The genetic distance of Cyt-b gene between Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia was ≤2.2％,and ranged from 8.9％ to 11.2％ with Citellus dauricus from Inner Mongolia.The Neighbor-Joining tree of COI,Cyt-b gene showed two major clusters.One of them were clustered by Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia,and another one was Citellus dauricus from Inner Mongolia.The Neighbor-Joining tree of COI gene showed that all samples from Shaanxi Province clustered in a group.In conclusion,the Spermophilus in Shaanxi Province were S.alaschanicus.

Assessing the mitochondrial DNA diversity of the Chagas disease vector Triatoma sordida (Hemiptera: Reduviidae)

Triatoma sordida is a species that transmits Trypanosoma cruzi to humans. In Brazil, T. sordida currently deserves special attention because of its wide distribution, tendency to invade domestic environments and vectorial competence. For the planning and execution of control protocols to be effective against Triatominae, they must consider its population structure. In this context, this study aimed to characterise the genetic variability of T. sordida populations collected in areas with persistent infestations from Minas Gerais, Brazil. Levels of genetic variation and population structure were determined in peridomestic T. sordida by sequencing a polymorphic region of the mitochondrial cytochrome b gene. Low nucleotide and haplotype diversity were observed for all 14 sampled areas; π values ranged from 0.002-0.006. Most obtained haplotypes occurred at low frequencies, and some were exclusive to only one of the studied populations. Interpopulation genetic diversity analysis revealed strong genetic structuring. Furthermore, the genetic variability of Brazilian populations is small compared to that of Argentinean and Bolivian specimens. The possible factors related to the reduced genetic variability and strong genetic structuring obtained for studied populations are discussed in this paper.

Comparison of the Cyt b Gene Sequences in Tibet Mini-Pig and Other Chinese Pigs / 中国实验动物学报

Objective To analyze the Cyt b gene sequences in Tibet mini-pigs and clarify the differences and genetic relationship with other Chinese pigs.Method The sequence of Cyt b gene was amplified from genome DNA of Tibet mini-pig,Bama miniature pigs,Guizhou xiang pigs and Wuzhishan (WZS) pigs.After sequencing,the base sequences were compared and analysed.The blood relationship tree and evolution position of Tibet mini-pig were established.Result There were 14 mutation sites between domestic pigs in China and pigs from Europe.Besides there was a significant differenee in two nucleotide site:a T→C switch in site 420 and the G→A switch in site 883 at the same time.Conclusion Chinese pigs include Bama miniature pigs,Guizhou xiang pigs and WZS pigs,have a very close blood relationship with some of Tibet mini-pigs.It has been confirmed that there is a certain genetic differentiation in the Tibet mini-pig.

Sequence and analysis of Cyt b gene of Agkistrodon and its adulterants sold in market / 中草药

Objective To differentiate the sequences of Cyt b gene Agkistrodon from its adulterants sold in the market in order to provide molecular evidence for identification of Agkistrodon. Methods Cyt b gene was used to sequence and analyze Agkistrodon and its adulterants sold in the market. Results There was a mixed phenomenon in Agkistrodon species. The differences of Cyt b gene sequence between Agkistrodon and its adulterants sold in the market are significant: the difference rates among the species of Agkistrodon are 0%—0.91%, the difference rates between Agkistrodon and its adulterants are 18.57%—23.78%. Conclusion The characteristics of Cyt b gene sequence can be used as a better molecular marker for authenticating Agkistrodon from its adulterants.