Rapid detection method of edwardsiella tarda
A slow-Edward, detection method technology, applied in the field of disease microorganism detection, can solve the problems of time-consuming, low sensitivity, and laborious, etc., and achieve the effects of improved efficiency, good specificity and repeatability, and high sensitivity
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Embodiment 1
[0036] Example 1: Design of primers and probes and optimization of reaction system
[0037] According to the gyrB gene sequence of Edwardsiella tarda (ATCC 15947) published in GeneBank, a pair of PCR amplification primers was designed (Table 1).
[0038] Table 1: Sequence Information Table of Related Primers and Probes
[0039]
[0040] The coding sequence of the gyrB gene of Edwardsiella tarda was obtained by PCR amplification and sequencing, and it was found that this sequence only had a high match with the genome of Listonia anguillarum by Blast comparison.
[0041] Search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) the highly conserved sequence region of the gyrB gene through the conserved structure domain, according to the principle of RPA amplification technology primer design, in the conserved region of the gyrB gene A probe was designed, and 5 upstream and 5 downstream RPA primers (ie F1, R1 in Table 1; F2, R2; F3, R3; F4, R4; F5, R5) were designed on bo...
Embodiment 2
[0044] (1) Extraction of bacterial genome
[0045] Listonella anguillarum ATCC19264, V.vulnificus ATCC27562, V.fluvialis LMG7894, Vibrioparahaemolyticus CGMCC1.1997, Vibrio harveyi harveyi CGMCC1.1599) these five kinds of Vibrio were streaked and inoculated in 2216E solid medium respectively, and after culturing at 37°C for 18 hours, a single colony was picked and placed in 2216E liquid medium, and cultured overnight in a shaker at 37°C until number of growing seasons;
[0046] Pseudomonas aeruginosa (CGMCC1.1785), Aeromonas hydrophila (CGMCC1.2017) and Pseudomonas fluorescens (CGMCC1.6279) were inoculated on LB solid medium by streaking, 30 After culturing at ℃ for 18 hours, pick a single colony and put it in LB liquid medium, and culture it overnight in a shaker at 30℃ until the logarithmic growth phase;
[0047]Edwardsiella tarda (E.tarda ATCC 15947), Edwardsiella catfish (E.ictaluriATCC33202), Edwardsiella baoke (E.hoshinae ATCC33379), Edwardsiella piscicida (E.piscicida...
Embodiment 3
[0050] Construction of LFD-RPA reaction system
[0051] Genomic DNA of Edwardsiella tarda (ATCC 15947) was used as the template of the LFD-RPA reaction system.
[0052] 1. Use the Basic-RPA reaction to screen out the primers with high amplification efficiency from F1, R1 to F5, R5 in Table 1.
[0053] Experimental group 1: 0.96 μL (10 μM) forward primer (F1), 0.96 μL (10 μM) reverse primer (R1), 11.8 μL buffer, 0.8 μL template (ie, genomic DNA of Edwardsiella tarda), plus ddH 2 0 to 19 μL, after mixing, add lyophilized enzyme powder, shake and mix, and centrifuge. Transfer the centrifuged premix to the RPA reaction tube, add 1.0 μL of starter MgAc (280mM), cover the lid, shake and centrifuge rapidly, so that the liquid on the tube wall is collected to the bottom of the tube.
[0054] Quickly transfer the dedicated reaction tube to the thermostatic metal bath with the programmed program. Reaction conditions: Incubate for 4 minutes at a constant temperature of 40°C, take out ...
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