Computational protocol: Molecular survey and characterization of a novel Anaplasma species closely related to Anaplasma capra in ticks, northwestern China

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Protocol publication

[…] Questing ticks were collected on the vegetation with the flagging method once a month between March and May 2011, in Gannan Tibetan Autonomous Prefecture (33°06"–36°10"N, 100°46"–104°44"E) in Gansu Province. The sampling sites were located in forest and pasturing areas that rely heavily on the farming of sheep, goats, and yaks for milk, and meat for the local economy. The average altitude at the sampling sites is over 3,000 m. Ticks were identified as Haemaphysalis qinghaiensis microscopically on the basis of morphological parameters []. DNA was extracted from adult H. qinghaiensis ticks individually using the Puregene DNA purification kit (Qiagen, Beijing, China) according to the manufacturer’s protocols.The DNA of 414 tick samples was screened for the presence of the gltA gene of Anaplasma sp. by nested PCR, with the primers and PCR conditions described in Table . The first-round PCR was carried out with previously published primers []; the primers for the second-round amplification and the primers targeting 16S rRNA and groEL genes were designed based on the corresponding sequences of A. capra HLJ-14 using Primer Premier 5.0 software (PREMIER Biosoft International, 3786 Corina way, Palo Alto, CA, USA) []. In order to further identifying the agent, the partial 16S rRNA and the groEL gene fragments were amplified from samples positive for gltA gene of the Anaplasma sp. (Table ). PCR reactions were performed in an automatic thermocycler (Bio-Rad, Hercules, USA). Genomic DNA extracted from infected ticks that had been verified by sequencing was used as the positive control, and sterile water was used as the negative control. PCR products were visualized by UV transillumination in a 1.0% agarose gel following electrophoresis and staining with ethidium bromide. The PCR products of the gltA (594 bp), 16S rRNA (1,261 bp) and groEL (874 bp) genes were purified (TaKaRa Agarose Gel DNA purification Kit Ver. 2.0, Dalian, China), cloned (pGEM-T Easy vector, Promega, Madison, WI, USA) and subjected to sequencing using BigDye Terminator Mix (Sangon, Shanghai, China). The GenBank accession numbers of Anaplasma sp. detected in H. qinghaiensis ticks in this study are as follows (not including identical sequences): KX673824 and KX673825 (16S rRNA), KX685885 and KX685886 (gltA) and KX685887 and KX685888 (groEL). Sequences were compared with the published sequences in GenBank by a BLASTn search and analyzed with the Clustal W method in the MegAlign software (DNAStar, Madison, WI, USA). Phylogenetic analysis was conducted based on the sequence distance method using the neighbor-joining (NJ) algorithm with the Kimura two-parameter model of the Mega 4.0 Software []. The results were analyzed using a Chi-square test in Predictive for Analytics Software Statistics 18 (PASW, SPSS Inc., Chicago, IL, USA). P-values of 0.05 or less were considered statistically significant. […]

Pipeline specifications

Software tools Primer Premier, BLASTN, Clustal W, MEGA
Application Phylogenetics
Organisms Escherichia coli, Capra hircus, Homo sapiens
Diseases Ataxia Telangiectasia