Computational protocol: Alicyclobacillus Contamination in the Production Line of Kiwi Products in China

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Protocol publication

[…] The sequence data of 16S rDNA of these isolates were compared to sequences in GenBank using BLAST 2.2.27 and then submitted to GenBank using Sequin. Then these 16S rDNA sequences of isolates were aligned with CLUSTAL W to construct a phylogenetic tree with MEGA 5.10 using both the neighbour-joining method based on the p-distance model and the maximum-parsimony method .RAPD-PCR amplifications were performed in 50 µL volume reactions containing 0.3 µL of 5 U/µL Taq DNA polymerase (Takara), 5 µL of 10× PCR reaction buffer, 3 µL of 25 mM MgCl2, 4 µL of dNTPs mixture (Takara) with 2.5 mM each, 4 µL of 2 different primers (A-01∶5′-CAGGCCCTTC-3′; AZ-14∶5′-CACGGCTTCC-3′) (BGI). The volume of template DNA was adjusted from 1 to 4 µL to get the clearest electrophoresis patterns. The PCR conditions were the same as above, except for annealing at 38°C for 45 s, and elongation at 72°C for 2 min 30 s. The results were checked by 1% agarose electrophoresis. […]

Pipeline specifications

Software tools Clustal W, MEGA
Databases RAPD
Application Phylogenetics
Chemicals Guaiacol