Urinary metagenomic sample library construction and detection method based on nanopore sequencing platform
A urinary metagenomic and nanopore sequencing technology, applied in biochemical equipment and methods, microbial measurement/testing, combinatorial chemistry, etc., can solve the problem of high proportion of human nucleic acid, affecting the accuracy of result output, and the amplification of infectious bacteria Opportunity does not wait for the problem to achieve the effect of improving the false negative problem of missing or less testing, improving the detection rate, and suitable for promotion
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Embodiment 1
[0077] Parameter optimization such as temperature and time of embodiment 1 library pcr
[0078] In view of the fact that RPB004 reagent is only a basic library building kit of ONT Company, and for the special sample of urine, its library building effect is not significant (such as the above-mentioned comparative example), the application for the above-mentioned comparative example in the research and development process The amplification system was repeatedly optimized for multiple factors, including pre-denaturation temperature, denaturation temperature, denaturation time, extension temperature and time, etc., and finally determined the ideal amplification parameter system.
[0079] Method: 0.1ng / 0.25ng / 0.5ng / 1ng of 4 gradients of the above standard and 1μl of FRM (components in the RPB004 kit) were added to each sample in a 0.2ml PCR tube to simulate the lower starting point in practical applications. initial situation. Gently mix and centrifuge, and react on the PCR instru...
Embodiment 2
[0090] DMSO introduction and concentration optimization of library pcr in embodiment 2
[0091] 1. Method A: Add 0.1ng / 0.25ng / 0.5ng / 1ng of the above-mentioned standards and 1μl FRM (purchased from ONT, included in the RPB004 kit) to each sample in a 0.2ml PCR tube for Mimics the case of lower starting amounts in real applications.
[0092] Prepare the PCR mix system (50 μl) as follows, the final concentration of DMSO is 2% (v / v):
[0093] Nuclease-free water 18μl Tagmented DNA 4μl RLB (01-12A) 1μl LongAmp Taq 2X master mix(NEB) 25μl 25% dimethyl sulfoxide DMSO 4μl
[0094] PCR instrument reaction, the conditions are as follows:
[0095]
[0096] Take 1 μl after PCR, and use Qubit to detect the concentration of PCR products, and the quality control standard is 4ng / μl.
[0097] Add an equivalent volume of 0.6×beads, rotate and incubate at room temperature for 5 minutes, centrifuge briefly and put it on a magnetic stand to remove th...
Embodiment 3
[0111] Embodiment 3 clinical sample detection
[0112] Microorganisms: clinical urine samples collected by the laboratory department of the hospital, each with clinical urine culture results. Samples were transported in a cold chain at 4°C, fully and gently mixed in a biological safety cabinet and centrifuged at 12300g for 5min, discarded the supernatant and added 1mL PBS to resuspend the pellet, and then transferred to a 2ml low adsorption centrifuge tube.
[0113] 1 to host:
[0114] a) Take the host sample that needs to be removed in the previous step, and centrifuge at 12300g for 5min.
[0115] b) Discard the supernatant carefully (do not touch the pellet, you can keep <=50μl supernatant), resuspend the pellet in 250μl PBS, vortex and mix well, if there is agglomerate, pipette up and down with a gun to mix well.
[0116] c) Saponin with a final concentration of 0.22% (w / v) was added, vortexed, and rotated on a rotary mixer at room temperature for 10 minutes. Add 350 μl ...
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