Microbial population analysis of tick salivary glands; a possible strategy for the surveillance of bacterial pathogens.
Adult ticks were collected using a flagging method in Shizuoka prefecture during 2008 to 2010. From each tick, a salivary gland was removed under a stereomicroscope, and genomic DNA was individually extracted by using QIAamp DNA Mini kit (QIAGEN, Tokyo, Japan). The V1 to V3 hyper-variable regions of bacterial 16S rDNA were amplified with PCR using universal primers as previously described: 27F and 518R. The 27F primer contained 10 bases of a multiplex identifier (MID) tag sequences. The PCR was performed in a total volume of 50 ?l containing 5 ?l of 10? PCR Buffer, 0.2 ?l of Platinum? Taq DNA polymerase (Life technologies, Tokyo, Japan ), 1 ?l of each primer, 1 ?l of 10 mM dNTP mixture, 1.5 ?l of 50 mM MgCl2, and 1 ?l of template DNA. The PCR conditions were initiated with a 2 min denature step at 94?C followed by 30 cycles of 94?C for 30 sec, 55?C for 30 sec, and 72?C for 1 min. The quality of the amplified PCR products (about 500 bp in length) were assessed by an electrophoresis in 1% agarose gel followed by the purification using the Wizard? SV Gel and PCR Clean-Up System (Promega, Tokyo, Japan). Subsequently, the concentration and quality of the amplified product were assessed on an Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, USA) using a DNA 1000 lab chip (Agilent Technologies). Each amplicon library with different MID tag were mixed together and subjected to 454 pyrosequencing Genome Sequencer Junior (GS Junior; Roche).