Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer and method for streptococcus equi subsp. Zooepidemicus and application
A real-time fluorescence quantification, Streptococcus equi technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, resistance to vector-borne diseases, etc., can solve the problems of low sensitivity, undetectable, time-consuming and costly, and achieve specificity Good results
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Embodiment 1
[0027] Example 1 Design and synthesis of specific amplification primer pairs
[0028] In this example, the pgm gene was amplified, and the analysis and alignment were performed according to the pgm gene sequence of SEZ published in GenBank (accession number: CP002904.1). Design a pair of specific primers (SEQ ID No. 1-2), upstream primer qPGM-F: 5'-CCCCGTAGCTCTCTGCAAT-3', downstream primer qPGM-R: 5'-GCAGACTGGGACGCTACC-3', the amplified fragment length is 89bp . The above primer pairs were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
Embodiment 2
[0029] The construction of embodiment 2 positive standard
[0030] In this example, the DNA of SEZ (C55138 strain was purchased from China Veterinary Drug Administration) was extracted with Mini BEST ViralRNA / DNA Extraction Kit, and its pgm gene was amplified by ordinary PCR. The reaction system is 25 μL: 12.5 μL of 2×PremixTap, 1.0 μL (10 μmol / L) of each of the upstream and downstream primers described in Example 1, 1.0 μL of template, ddH 2 O 7.5 μL. The amplification program was 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min; and 72 °C for 5 min. PCR amplification products were electrophoresed on 1.0% agarose. Then, according to the instructions of the DNA Purification Kit, the PCR product was recovered and purified, and the purified PCR product was ligated with the pMD19-T vector through Solution I ligase, and then the ligated product was thermally transformed into DH5α E. coli competent cells. Amp+ The positive clone obtained by plate scr...
Embodiment 3
[0032] Example 3 Establishment of the standard curve of fluorescence quantitative PCR
[0033] The recombinant plasmid pMD19T-SEZ was serially diluted to obtain 3.87×10 2 copies / μL~3.87×10 9 copies / μL of 8 dilutions of the standard as the reaction template. Three replicates were set for each dilution gradient and a negative control was established. Fluorescence quantitative PCR reaction system is 20 μL: SYBR GreenⅡMix (2×) 10 μL, upstream and downstream primers (SEQID No.1-2) each 0.6 μL (10 μmol / L), template 2 μL, ddH 2 O 6.8 μL. The reaction conditions were: (1) Pre-denaturation: 95°C, 30s, 4.4°C / s, 1 cycle; (2) PCR reaction: 95°C, 5s, 4.4°C / s; 54°C, 30s, 2.2°C / s; 72°C, 30s, 4.4°C / s, a total of 40 cycles. (3) Dissolution curve: 95°C, 5s, 4.4°C / s; 60°C, 1min, 2.2°C / s; 95°C, 0s, 0.11°C / s.
[0034] Taking the logarithm of the copy number of the standard as the X-axis and the ΔCT value as the Y-axis, draw the standard curve as follows figure 2 As shown, the standard curv...
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