Method for rapid identification of Edwardsiella fisicida sRNA

[0035] Embodiment 2 real-time fluorescent quantitative PCR analysis

[0036] In this example, real-time fluorescent quantitative PCR was used to analyze the expression difference of sRNA082 between the wild strain and the mutant strain.

[0037] Methods: The wild strain of Edwardsiella piscicida and the hfq mutant strain were cultured to the logarithmic phase (OD=0.8), the bacteria were collected, and the RNA was extracted with the EZNA Total RNA Extraction Kit from Omega Company, and the RNA was treated with RNase-free DNaseI. Take 1 μg of RNA sample, and use Invitrogen’s Superscript II reverse transcriptase to perform reverse synthesis of cDNA; use cDNA as a template for real-time fluorescent quantitative PCR: the reagent is Takara PrimeScript TM RT-PCR KitII; reaction parameters: pre-denaturation at 95°C for 30s, denaturation at 95°C for 10s, annealing and extension at 60°C for 30s, 40 cycles; sRNA primers (taking Edwardsiella ichthyophila sRNA012 as an example), 082RTF:AA...

[0039] Embodiment 3 Accuracy detection test

[0040] Using the method of Example 1 of the present invention and the real-time fluorescent quantitative PCR experiment conditions of Example 2, 19 sRNA samples were identified, among which, 19 samples were determined to be Hfq-dependent sRNA by transcriptome sequencing. see results figure 2 .

[0041] The results show that the method provided by the present invention detects the above 19 samples, and determines that 16 samples are Hfq-dependent sRNA, and three samples of sR063, sR009, and sR162 are Hfq-dependent sRNA, with an accuracy rate of 84.2%. It shows that the method provided by the invention can quickly and accurately identify the dependence relationship between sRNA and Hfq protein.