Sterility detection method based on NGS technology
A sequencing and genome technology, applied in the field of bioinformatics, can solve the problems of long cycle time and low sensitivity, and achieve the effect of small amplification bias, high amplification sensitivity and fast speed
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Embodiment 1
[0118] Embodiment 1 Carry out the whole genome amplification of microbial trace DNA based on MDA method
[0119]In order to compare the use of different microbial lysis methods, this implementation case uses 4 μL of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes, and Candida albicans containing 100 CFU respectively. 4 μL of Nuclease-free water was used as a negative control, according to method 1: add 3 μL lysozyme and treat at 65°C for 10 minutes; method 2: treat at 90°C for 10 minutes; method 3: add lysozyme and treat at 65°C for 10 minutes, then treat at 90°C 5 minutes. After the samples have been processed by different methods, briefly centrifuge to collect the tube wall liquid to the bottom of the tube, add 40 μL of the amplification reaction mixture (Amplification Reaction Buffer 39 μL, Amplification Enzyme 1 μL) of the MGIEasy Single Cell Whole Genome Amplification Kit manufactured by MGI, and shake Mix well. ...
Embodiment 2
[0121] The sensitivity test of embodiment 2 microbial sample detection
[0122] A total of 6 bacteria and one fungus (Fusobacterium nucleatum, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes, Candida albicans) were taken, and each microorganism set 5 The number of strains of each gradient, the number of microorganisms correspond to 1CFU, 5CFU, 10CFU, 50CFU, 100CFU and negative control group (sterile water) respectively, and the scheme is shown in Table 1. After the DNA of each sample was amplified by MDA, high-throughput sequencing was performed by the DNBSEQ-T7 sequencing system. The sequencing strategy used PE100 (that is, paired-end sequencing, the length of each read segment was 100bp), and the amount of sequencing data for each sample was not less than 1Gb. Obtain sequencing data for each sample.
[0123] Table 1 The detection sensitivity test of different single bacterial species (the brackets after the bacteria...
Embodiment 3
[0132] Example 3 Mixed microorganism NGS method accuracy detection test
[0133] In order to test the detection sensitivity of microorganisms in the mixture of different microorganisms, Escherichia coli and Staphylococcus aureus were mixed according to different proportions, and DNA extraction, MDA amplification and sequencing were performed on the mixed microorganisms, and the amount of sequencing data was not large. Less than 1G, the data analysis process and software parameters are consistent with the analysis process of single microorganism sensitivity detection in Example 2. The specific experimental plan is as follows:
[0134] Table 2 Detection sensitivity test of different mixed strains (the brackets after the strains are the codes of the bacteria)
[0135] bacteria sample plan Escherichia coli (D), Staphylococcus aureus (J) DJ-0 D and J are mixed according to 100 to 0 CFU Escherichia coli (D), Staphylococcus aureus (J) DJ-0.1 D and J a...
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