Chimeric multiplex PCR primer composition and detection method
A primer composition, chimera technology, applied in biochemical equipment and methods, recombinant DNA technology, microorganism determination/inspection, etc., can solve the problems of low detection sensitivity and large sensitivity fluctuations, etc.
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Embodiment 1
[0079] Genomic DNA was extracted from the blood of two healthy volunteers (as a donor and a recipient, respectively) using a whole blood genomic DNA extraction kit, cat: 51185, Qiagen.
[0080]The two genomic DNAs obtained above were quantified using ThermoFisher's Qubit3.0 quantifier (cat: Q33218), and mixed according to the ratios of 9:1, 99:1, 995:5, 999:1 and 9995:5, As test samples 1, 2, 3, 4 and 5, chimeric DNA was simulated.
[0081] The unlabeled primers (primer pairs 1-48) in Table 1 were synthesized by chemical synthesis. After the concentration of each primer was diluted to 10 μmol / L, all 48 pairs of primers were mixed in equal volumes and diluted with deionized water to the primer Final concentration is 100nmol / L, and this mixture is the PrimerMix that uses subsequently.
[0082] multiplex PCR amplification
[0083] Using a 0.2ml PCR tube, configure the PCR reaction in a clean bench according to the following system: Thermo Fisher High-Fidelity PCR Amplification ...
Embodiment 2
[0099] Genomic DNA was extracted from the blood of two healthy volunteers (as a donor and a recipient, respectively) using a whole blood genomic DNA extraction kit, cat: 51185, Qiagen.
[0100] The two genomic DNAs obtained above were quantified using ThermoFisher's Qubit3.0 quantifier (cat: Q33218), and mixed according to the ratios of 9:1, 99:1, 995:5, 999:1 and 9995:5, As test samples 1, 2, 3, 4 and 5, chimeric DNA was simulated.
[0101] The unlabeled primers (primer pairs 1-25) in Table 1 were synthesized by chemical synthesis method, and each of the 25 pairs of primers was diluted to 10 μmol / L and then mixed in equal volumes, and the final concentration of the primers was diluted with deionized water. 100nmol / L, this mixture is the PrimerMix used subsequently.
[0102] multiplex PCR amplification
[0103] Using a 0.2ml PCR tube, configure the PCR reaction in a clean bench according to the following system: Thermo Fisher High-Fidelity PCR Amplification Reagent 15μl, Pri...
Embodiment 3
[0119] Genomic DNA was extracted from the blood of two healthy volunteers (as a donor and a recipient, respectively) using a whole blood genomic DNA extraction kit, cat: 51185, Qiagen.
[0120] The two genomic DNAs obtained above were quantified using ThermoFisher's Qubit3.0 quantifier (cat: Q33218), according to the ratios of 9:1, 99:1, 995:5, 999:1, 9995:5 and 9999:1, respectively. The ratios were mixed as test samples 1, 2, 3, 4, 5 and 6, simulating chimeric DNA.
[0121] The same detection steps as in Example 1 were used for chimera detection, and the multiplex PCR primers used were identification primer pairs 49-96. Each primer in the 48 pairs of primers was diluted to 10 μmol / L and mixed according to equal volumes, and deionized water The final concentration of diluted primers is 100nmol / L, and this mixture is the PrimerMix used subsequently.
[0122] Multiple PCR primer sets used for the same test sample use the same index primers. In this experiment, the index primers u...
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