A liquid-phase chip kit for serotype typing of Streptococcus suis
A liquid phase chip, Streptococcus suis technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high cost, inaccurate results, large workload, etc., and achieve short detection period and sensitivity. High, short detection time effect
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Embodiment 1
[0028] Embodiment 1 kit design principle
[0029] The present invention first designs 20 specific primers for S. suis specific serotype typing from NCBI-GeneBank according to the differences in the capsular gene (Cps Gene) of different serotypes of Streptococcus suis. SEQ ID NO: 1~ 40; 20 serotypes of Streptococcus suis were divided into four groups of multiplex PCR systems on the premise of ensuring primer specificity and no dimer influence (standard strains donated by OIE Streptococcus Reference Laboratory, see Table 1 for details), p. One group is serotypes 1, 2, 3, 7, 9, 11, 16, see figure 2 ; the second group is serotypes 5, 8, 12, 19, 25, see image 3 ; the third group is serotypes 21, 27, 28, 30, see Figure 4 ; the fourth group is serotypes 13, 15, 23, 31, see Figure 4 ; Add a TAG sequence to the 5' end of the specific upstream primer, use 12 carbon bridges as a termination signal in the middle, and add Biotin to the 5' end of the specific downstream primer, see S...
Embodiment 2
[0033] Embodiment 2 kit composition
[0034] Multiplex PCR reagent: PCR system 24.5μL
[0035] Combination 1 (contains 12.5 μL of multiplex PCR Mix, 0.8 μL each of primers for serotypes 1, 2, 3, 7, 9, 11, and 16, ddH 2O to 24.5 μL), combination 2 (including 12.5 μL of multiplex PCR Mix, 0.8 μL of primers for serotypes 5, 8, 12, 19, and 25, ddH 2 O to 24.5 μL), combination 3 (including 12.5 μL of multiplex PCR Mix, 0.8 μL of primers for serotypes 21, 27, 28, and 30, ddH 2 O to 24.5 μL), combination 4 (including 12.5 μL of multiplex PCR Mix, 0.8 μL of primers for serotypes 13, 15, 23, and 31, ddH 2 O to 24.5 μL).
[0036] Liquid phase chip reagent: 90 μL of reaction system, including 1 μL of each coded reaction microsphere, 1×Tm to 20 μL, 70 μL of SAPE diluent (69.3 μL of 1×Tm and 0.7 μL of SAPE).
Embodiment 3
[0037] Example 3 Kit usage method
[0038] 1. Multiplex PCR
[0039] The reaction system is 25 μL, 24.5 μL multiplex PCR reagents (combinations 1, 2, 3, 4) and 0.5 μL nucleic acid to be tested (10 ng / μL).
[0040] Operation steps: After diluting the nucleic acid of the sample to be tested according to the above requirements, add it to the 4 combined multiplex PCRs in the kit.
[0041] Reaction conditions: 95°C for 3min; 95°C for 20s; 60°C for 1min; 72°C for 90s; 35 cycles, 72°C for 10min.
[0042] 2. Hybridization
[0043] 1. Use 2500 microspheres for each coded microsphere to participate in the reaction (ie 1 μL), and make up to 20 μL with 1×Tm;
[0044] 2. Add 2 μL of the above-mentioned PCR amplified product, and use ddH 2 O was adjusted to a final volume of 25 μL;
[0045] 3. Dilute SAPE to 8 μg / ml with 1×Tm hybridization buffer, add 70 μL to each reaction well, and mix gently;
[0046] 4. Put it into the PCR instrument and hybridize for 35 minutes at 40°C (see Fig...
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