A kind of pathogenic microorganism detection method and kit based on metagenomics
A pathogenic microorganism and metagenomics technology, applied in the field of pathogenic microorganism detection methods and kits based on metagenomics
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experiment example 1
[0096] In this example, an experiment is carried out on the reaction system of a fragmentation enzyme, and the fragmentation enzyme is commercially available DNA Fragmentase.
[0097] plan a
[0098] DNA sample 1 μl, fragmentation buffer 1 μl, fragmentation enzyme 2 μl, Enhance 0.5 μl, Buffer EB to make up the system to 15 μl,
[0099]
[0100] Option b
[0101] DNA sample 1 μl, fragmentation buffer 2 μl, fragmentation enzyme 4 μl, Enhance 1 μl, Buffer EB to make up the system to 15 μl, and the reaction conditions refer to scheme a.
[0102] plan c
[0103] DNA sample 1 μl, fragmentation buffer 1.5 μl, fragmentation enzyme 3 μl, Enhance 0.75 μl, Buffer EB to make up the system to 15 μl, and the reaction conditions refer to scheme a.
[0104] plan d
[0105] DNA sample 1 μl, fragmentation buffer 1.5 μl, fragmentation enzyme 5 μl, Enhance 1 μl, Buffer EB to make up the system to 15 μl, and the reaction conditions refer to scheme a.
[0106] Scheme e
[0107] DNA sample ...
experiment example 2
[0128] Since polyvinylpyrrolidone K30 (PVP-K30) has a certain cross-linking property at a certain content as an excipient of pharmaceutical preparations, the applicant believes that this cross-linking property may have a positive effect on promoting the uniformity of the fragmentation reaction.
[0129] Therefore, based on the scheme b in Experimental Example 1, 0.02 mg of PVP-K30 was added to the system to carry out the fragmentation reaction experiment. The results are shown in Table 4:
[0130] Table 4
[0131]
[0132]
[0133] However, compared with the results in Table 2, the results in Table 4 show that the homogeneity after the fragmentation reaction is not improved, but slightly decreased.
[0134] In subsequent experiments, the applicant continued to use scheme b as the basis, adding 0.2 μl polyethylene glycol 2000 and 0.02 mg of PVP-K30 to the system, and carried out fragmentation reaction experiments. The results are shown in Table 5:
[0135] table 5
[01...
experiment example 3
[0139] In this example, experiments were carried out on the scheme of magnetic bead purification.
[0140] Sample 1: Take the product of Scheme b in Example 1 as Sample 1.
[0141] Sample 2: Based on scheme b in Example 1, 0.2 μl of polyethylene glycol 2000 was added to the system, and the reaction product was sample 2.
[0142] Scenario A, A':
[0143] S1, pipette 10 μL EBBuffer and 30 μL DNA Clean Beads into 20 μL sample, and mix well;
[0144] S2, incubate at room temperature for 1 min;
[0145] S3, after centrifugation, separate the magnetic beads and the liquid under the action of an external magnetic field, and remove the supernatant after the solution is clarified;
[0146] S4, continue to add 100 μL of 70% ethanol solution to rinse the magnetic beads under the action of an external magnetic field, incubate at room temperature for 10 seconds, and remove the supernatant;
[0147] S5, exposing the magnetic beads to air for 5 minutes to dry,
[0148] S6, add 10 μL of ...
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