A primer pair, kit and method for detecting and identifying bacteria belonging to the genus Dickia
A Dick's genus and primer pair technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., and can solve the problems of easy detection, low specificity and low versatility.
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Embodiment 1
[0042] Design universal specific primers for Dickeya bacteria according to the unique gene sequence of Dickeya bacteria:
[0043] Whole genome sequences of bacteria of the family Pectobacteriaceae were downloaded from the NCBI Genome Database. For the whole genome sequences of the same strain measured by different institutions or different methods, select sequences with high quality sequencing and assembly, ContigN50 is greater than 50,000, and the pseudogene rate is less than 10%; Whole genome sequences with integrity greater than 95% and contamination less than 5% were selected for pan-genomics analysis. The unique proteins of Dickeya were found from the Orthologs_Cluster results obtained by the pan-genome analysis, and then the amino acid sequences of the unique proteins were searched using the NR database in NCBI, and the proteins with homologous sequences in other strains of the family and genus were excluded. For the nucleotide sequence of the unique protein of Dickeya ...
Embodiment 2
[0059] The sensitivity of the primer pairs to detect bacteria of the genus Dickeya was verified.
[0060] The genomic DNA of Dickeya dadantii strain CZ1501 with a concentration of 100ng / μL was serially diluted by 10 times, and 1 μL of DNA solution of each dilution was used as a template, and primers DicCSP-F1 or DicCSP-F2 and DicCSP-R1 or DicCSP-R2 were used in In a 25μL reaction system, 2×Hieff PCPMaster Mix produced by Shanghai Yisheng Biotechnology Co., Ltd. was used to carry out PCR with the above amplification procedure, and the PCR product (123bp) was detected by agarose gel electrophoresis. The lowest concentration of detectable DNA was 10pgμL -1 (by image 3 to represent).
Embodiment 3
[0062] Fluorescence quantitative PCR was performed with primer pair DicCSP-F / DicCSP-R to detect the bacteria of the genus Dickya.
[0063] The genomic DNA of Dickeya dadantii strain CZ1501 with a concentration of 100ng / pL was serially diluted in 8 dilutions by 10 times, and 1 μL of DNA solution was taken as the template. Primer DicCSP-R1 (10 μM), 10 μL of 2×T5 Fast qPCR Mix, 9.2 μL of sterile ultrapure water, using Bio-RadCFX-96 real-time fluorescence quantitative PCR instrument, with the amplification program (95 °C pre-denaturation for 1 min; 95 °C denaturation 10s, annealing at 60°C for 5s, extension at 72°C for 15s, 40 cycles) for quantitative PCR.
[0064] The minimum DNA concentration positive for amplification is 100 fg μL -1 ( Figure 4 ), which is more sensitive than conventional PCR for DNA detection (10pgμL -1 ) 100 times higher. The melting curve of the amplification result has one and only one peak ( Figure 5 ), indicating that the amplification product is s...
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