Method for detection and differential diagnosis of Brucella in aerosol
A technology of Brucella and differential diagnosis, applied in the field of detection and differential diagnosis of Brucella in aerosols of farms
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Embodiment 1
[0054] The design and synthesis of embodiment 1 primer
[0055] The comparison of the VirB10 gene sequence of bovine (GenBank: AF226278.1, sequence 10 in the sequence listing), sheep (GenBank: DQ861303.1) and pig (GenBank: AF141604.1) found that it is highly conserved, see figure 1 . According to the conserved sequence of Brucella bovis VirB10 gene in GeneBank, design full-length amplification primer (as shown in SEQIDNO.1,2), be used for the construction of positive standard plasmid (sequence 1,2 in table 1); Adopt PrimerExpress3 .0 software, design a pair of specific primers (as shown in SEQIDNO.3,4), be used for Brucella universal detection (sequence 3,4 in table 1). According to the Brucella bovis AlkB gene and the IS711 sequence (GenBank: AF148682.1, shown in SEQ ID NO.11) respectively in GeneBank, the Brucella caprica insertion sequence IS711 (GenBank: JF939171.1, shown in SEQ ID NO.12 Shown), the chromosomal sequence of Brucella suis S1330 strain (GenBank: 1618935~161...
Embodiment 2
[0058] The establishment of Brucella passing type detection method in embodiment 2 aerosol
[0059] (1) Establishment of SYBRGreenⅠreal-time fluorescent quantitative PCR method
[0060] 1. Preparation of samples to be tested
[0061] Genomic DNA was extracted according to the instructions of the bacterial DNA extraction kit.
[0062] 2. Preparation of standard products
[0063] Carry out PCR amplification with the DNA of the Brucella bovis live vaccine (A19 strain) that step 1 obtains as template, and reaction system is 50 μ L: 10 * LAPCRBufferII (Mg 2+ Plus) 5 μL, 2.5mMdNTPMixture8 μL, LATaq0.5 μL (5U / μL), template 2 μL, 10 μmol / L of VirB10 gene full-length upstream and downstream primers VirB10-1 (sequence 1 and sequence 2 in Table 1) each 2 μL, sterilized supernatant Add pure water to 50 μL. The reaction program was pre-denaturation at 94°C for 3min, followed by 30 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 1min; extension at 72°C for 10min, and finally stopped ...
Embodiment 3
[0085] Establishment of method for differential diagnosis of bovine species, sheep species and swine brucella in embodiment 3 aerosol
[0086] Respectively with the DNA of the Brucella bovis live vaccine A19 strain, the Brucella sheep species live vaccine M5-90 strain and the Brucella suis live vaccine S2 strain as a template, simultaneously use Abortus primers (such as SEQ ID NO.5 , shown in 6), Melitensis primer (as shown in SEQIDNO.6,7) and Suis primer (as shown in SEQIDNO.8,9), carry out primer concentration, annealing according to 3 and 4 of step (1) in embodiment 2 The optimization of temperature and reaction condition, other methods are with embodiment 2. application The TmCalling analysis method of 480II GeneScanningSoftwareVersion1.5 calculates the Tm values of Brucella bovine, sheep and porcine species respectively, and makes differential diagnosis through the difference of Tm values. The results are as follows Figure 8 , 9 , 10, one specific melting curve wa...
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