Quantifying Gut Bacteria in Sand Flies

The gut bacteria in sand flies were quantified by qPCR with TB Green Fast Mix (TOYOBO Co., Ltd., Osaka, Japan) using a Thermal Cycler Dice Real Time System Lite (Takara Bio Inc., Shiga, Japan). The bacterial 16S rRNA gene was used as the target, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference. The primer sequences were 5ʹ-ACHCCTACGGGDGGCWGCAG-3ʹ (16S-q-337F) and 5ʹ-GTDTYACCGCGGYTGCTGGCAC-3ʹ (16S-q-514R) for the amplification of bacterial 16S rRNA gene, and 5ʹ-TTCGCAGAAGACAGTGATGG-3ʹ (Lugapdh-q-F) and 5ʹ-CCCTTCATCGGTCTGGACTA-3ʹ (Lugapdh-q-R), and 5ʹ-CGACTTCAACAGCA ACTCCCACTCTTCC-3ʹ (Phgapdh-q-F) and 5ʹ-TGGGTGGTCCAGGGTTTCTTACT CCTT-3ʹ (Phgapdh-q-R) for gapdh gene [35 , 36 (link)]. Relative quantities of 16S rRNA genes were determined by ∆∆Ct method using GAPDH as the reference.

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Tabbabi A., Mizushima D., Yamamoto D.S, & Kato H. (2023). Effects of host species on microbiota composition in Phlebotomus and Lutzomyia sand flies. Parasites & Vectors, 16, 310.

Publication 2023

Thermal Cycler Dice Real Time System Lite

16s rrna Bacterial Bacterial gene Gapdh Gene amplification Genes Glyceraldehyde 3 phosphate dehydrogenase Primer Q gene Sand flies

Corresponding Organization :

Other organizations : Jichi Medical University

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Variable analysis

independent variables

Gut bacteria in sand flies

dependent variables

Quantity of gut bacteria in sand flies measured by qPCR

control variables

Use of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene for relative quantification of 16S rRNA genes

controls

Positive control: Not specified
Negative control: Not specified

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