2.2. Amplicon sequencing of the bacterial 16S rRNA gene and fungal internal transcribed spacer region

In order to determine potential changes in microbiota compositions, larvae samples from AFB-infected apiary (group AFB and group CT.AFB) and CBD-infected apiary (group CBD and group CT.CBD) were subjected to amplicon sequencing of the 16S rRNA gene and internal transcribed spacer (ITS)1 region, respectively. Foragers from group DIS and group CT.DIS were subjected to both 16S rRNA gene and ITS1 region amplicons sequencing. Sequencing of this study was performed at Novogene Biological Information Technology Co., Ltd, Beijing, China.

A previously described CTAB/phenol-based extraction protocol [21] was used to extract total genomic DNA (gDNA) from larvae/forager samples. To this end, forager samples were prepared as follows. Individual forager was first rinsed in ample sterile water three times to remove the microorganisms on its body surface. Then, three foragers per colony were pooled together in 50 ml sterile tubes and homogenized with sterilized distilled H2O (1%, w/v) using a homogenizer. The obtained homogeneous solution was filtered through sterilized one-layer gauze to remove the floating slimy materials and then centrifuged at 3500g for 25 min. The resulting pellets, which contained microbes from the guts and other parts of honeybees, were collected for further DNA extraction.

Primer pairs (515F: 5′-GTGCCAGCMGCCGCGGTAA-3′; 806R: 5′-GGACTACHVGGGTWTCTAAT-3′) and (ITS5-1737F: 5′-GGAAGTAAAAGTCGTAACAAGG-3′; ITS2-2043-R: 5′-GCTGCGTTCTTCATCGATGC-3′) were used to amplify the hyper-variable V4 region of the 16S rRNA gene of bacteria and the ITS1 region of fungi, respectively. PCR amplifications were conducted in a total reaction volume of 25 µl, using 12.5 µl of Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, Beijing, China), 2.5 µl of each primer (5 µM) and approximately 10 ng of template gDNA. The procedures for PCR amplification were as follows: an initial denaturation step at 98°C for 2 min, followed by 25 cycles of 98°C for 30 s, 55°C for 30 s and 72°C for 30 s, ended with a final extension step at 72°C for 5 min. Amplicons were visualized by 2% agarose gel electrophoresis and purified using a Thermo GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Shanghai, China). The sequencing library was constructed by using Ion Plus Fragment Library Kit (48 rxns, Thermo Fisher Scientific) according to the manufacturer's instructions. After being quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and the assessment of the size on an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA), libraries were pooled in equimolar amounts and subjected to sequencing on an Ion S5TMXL (Thermo Fisher Scientific) platform according to standard protocols.

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