Double real-time fluorescent quantitative pcr identification method of brucella s2 vaccine strain and the kit of reagents used
A Brucella and non-Brucella technology, applied in the biological field, can solve the problem that immune antibodies cannot be distinguished from natural infection antibodies, and achieve the goals of preventing large-scale outbreaks, shortening detection time, ensuring development and public health safety Effect
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Embodiment 1
[0065] Embodiment 1, be used for the preparation of the test kit identifying Brucella S2 vaccine strain
[0066] 1. Screening of a complete set of reagents for identifying Brucella S2 vaccine strains
[0067] 1. Through a large number of sequence acquisition, analysis, comparison and preliminary experiments, the inventor of the present invention finally takes the IS711 gene in the Brucella genome as the target gene, screens and synthesizes a combination of primers and probes for detecting Brucella Bru-F1 / Bru-R1 / Bru-Probe1; against deletions specific to the S2 vaccine strain (e.g. figure 1 shown), design and synthesize the primer-probe combinations S2-F1 / S2-R1 / S2-Probe1, S2-F2 / S2-R2 / S2-Probe2 and S2-F3 / for identifying Brucella S2 vaccine strains S2-R3 / S2-Probe3. Perform Bru-F1 / Bru-R1 / Bru-Probe1 with S2-F1 / S2-R1 / S2-Probe1, S2-F2 / S2-R2 / S2-Probe2 and S2-F3 / S2-R3 / S2-Probe3 respectively Combining, the set of reagents 1, set of reagents 2 and set of reagents 3 are obtained in seq...
Embodiment 2
[0077] Embodiment 2, establishment of the method for identifying Brucella S2 vaccine strain by double real-time fluorescent quantitative PCR
[0078] 1. Drawing of standard curve
[0079] 1. Refer to the whole genome sequence of Brucella S2 vaccine strain, Brucella A19 vaccine strain, Brucella melis 16M strain, and Brucella bovis 2308 strain in the NCBI database, provided by Yingweijieji Trading Co., Ltd. Co., Ltd. artificially synthesized plasmid IS711 and plasmid S2.
[0080] Plasmid IS711 contains the nucleotide sequence of Brucella IS711 gene. The nucleotide sequence of the Brucella IS711 gene is shown in SEQ ID NO:10.
[0081] Plasmid S2 contains specific DNA sequences. The nucleotide sequence of the specific DNA sequence is shown in SEQ ID NO:11.
[0082] The Brucella S2 vaccine strain does not contain this specific DNA sequence, but other Brucella bacteria contain this specific DNA sequence.
[0083] 2. Detect the concentration of plasmid IS711 and plasmid S2 with ...
Embodiment 3
[0110] Embodiment 3, specific detection
[0111] 1. According to the method of step 3 in Example 2, specificity detection is carried out. The samples to be tested are Brucella S2 vaccine strain, Brucella A19 vaccine strain, Brucella ovis 16M strain, Brucella bovis 2308 strain, Brucella suis 1330 strain, dog cloth Rutella strain 6 / 66, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O:157 and Yersinia O:9.
[0112] See some results Figure 4 (A is the Brucella A19 vaccine strain, B is the Brucella S2 vaccine strain). The results showed that Brucella A19 vaccine strain, Brucella melis 16M strain, Brucella bovis 2308 strains, Brucella suis 1330 strains and Brucella canis 6 / 66 strains (all Brucella, which is a non-Brucella S2 vaccine strain), has two "S"-shaped amplification curves, Brucella S2 vaccine strain has one "S"-shaped amplification curve, Staphylococcus aureus, Salmonella typhimurium, E. coli O:157, Yersinia O:9, and the negative control all had no "S"...
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