Computational protocol: Seroepidemiological Survey of Zoonotic Diseases in Small Mammals with PCR Detection of Orientia tsutsugamushi in Chiggers, Gwangju, Korea

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Protocol publication

[…] The method used by Ree et al. [] was applied for the detection of O. tsutsugamushi from individual chiggers. Individual chiggers were placed on a glass slide with PBS 10 μl. The chigger’s internal contents were squeezed out with 2 fine pins and observed after suspension with 50 μl PBS under stereomicroscope (Carl Zeiss). The chigger exoskeleton was mounted with polyvinylalcohol medium (Bioquip, Gardena, California, USA) and identified by Ree’s fauna key []. Because it was difficult to perform with all chiggers, 30 chiggers per small mammals were used.DNA was extracted from 20 μl of chigger suspension using G-spin total DNA extraction kit (cat. no. 17046; Intron Biotechnology, Seoul, Korea). 200 μl of lysis buffer and 10 μl of proteinase K solution (20 mg/ml) was added to 20 μl of chigger suspension. The lysate was incubated at 56˚C on a heating block for 30 min. After lysis, 200 μl of binding buffer was added. Then, the mixture was incubated at 70˚C for 5 min. The mixture was applied to the spin column, and centrifuged at 13,000 rpm for 1 min. After discarding the filtrate, washing buffer A was added to the spin column and centrifuged at 13,000 rpm for 1 min. After discarding the flow-through, the column was placed into a 2.0 ml collection tube. Washing buffer B was added to the spin column and centrifuged at 13,000 rpm for 1 min. After discarding the flow-through, placed column into a new 1.5 ml collection tube, and a total of 50 μl of elution buffer was added directly onto the membrane. After incubating for 1 min at room temperature, it was centrifuged for 1 min at 13,000 rpm to elute. DNA extract was stored at -20˚C until amplification.PCR was performed as INNOPLEX TSUTSU detection kit (cat. No. IPC10040; Intron Biotechnology). The kit was designed using primer sets to detect the 475 bp fragment gene encoding the 56 kDa antigen of O. tsutsugamushi. The first PCR was performed with 2 μl of DNA extract and 18 μl of distilled water treated with diehyl pyrocarbonate (DEPC; Gendepot, Barker, Texas, USA) in the first PCR premix tube. PCR conditions were as follows: initial denaturation at 94˚C for 5 min; 40 cycle at 94˚C for 30 sec, 58˚C for 30 sec, 72˚C for 40 sec; and final elongation at 72˚C for 5 min using a Geneamp 9700 Biosystem (ABI, Foster City, California, USA). The second PCR was performed with 2 μl of the first PCR product and 18 μl of distilled water with DEPC in the second PCR premix tube. PCR conditions were as follows: initial denaturation at 94˚C for 5 min; 30 cycle at 94˚C for 30 sec, 58˚C for 30 sec, 72˚C for 40 sec, and final elongation at 72˚C for 5 min using a Geneamp 9700 Biosystem (ABI). The final PCR products were evaluated by 1.5% agarose gel electrophoresis containing ethidium bromide.The nucleotide sequence homology was aligned with sequence of previously published O. tsutsugamushi in Genbank by the BIOEDIT software program. The phylogenetic tree was generated by the neighbor-joining method (MEGA 6.0). […]

Pipeline specifications

Software tools BioEdit, MEGA
Application Phylogenetics
Organisms Orientia tsutsugamushi, Apodemus agrarius, Myodes regulus, Crocidura lasiura, Leptotrombidium pallidum
Diseases Zoonoses