Application of tuberculosis microbial marker in preparation of reagents for diagnosing tuberculosis
A technology for tuberculosis and microorganisms, which is applied in the field of application of tuberculosis microbial markers in the preparation of reagents for diagnosing tuberculosis, and can solve problems such as no research on tuberculosis intestinal flora biomarkers
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Embodiment 1
[0073] Example 1: Sample collection
[0074] All 20 stool samples were collected from 20 volunteers respectively, and the stool sample collection was performed by the Sixth People's Hospital of Dongguan, China. Diagnosed tuberculosis patients were used as the case group, and other non-diabetic individuals were used as the control group.
Embodiment 2
[0075] Example 2: DNA extraction and sequencing
[0076] 2.1 Storage of stool samples
[0077] Put the collected stool samples into sterilized stool collection tubes, store them in the freezer immediately, and add glycerol to the stool samples to freeze. Frozen samples were sent to the storage point and stored at -80°C until use.
[0078] 2.2 DNA extraction
[0079] 200 mg of frozen fecal samples were taken from each part and extracted with QIAGEN Fecal DNA Extraction Kit.
Embodiment 3
[0080] Example 3: Identification of biomarkers
[0081] refer to figure 1 and figure 2 The experimental process for the determination and identification of tuberculosis biomarkers.
[0082] 3.1 Basic processing of sequencing data
[0083] EBI raw data has been quality controlled and de-hosted, and the data processing steps are as follows:
[0084] (1) Remove adapter-contaminated Reads (the number of adapter-contaminated bases in Reads is greater than 5 bp);
[0085] (2) Remove low-quality Reads (more than 15% of Reads with a base quality value lower than 19);
[0086] (3) Remove the Reads containing more than 5% N.
[0087] 3.2 Obtaining TB microbiome gene sets
[0088] The main body of metagenomic biomarkers is genes and corresponding functions, so it is necessary to assemble sequenced sequences and predict genes, remove redundancy, and construct a non-redundant reference gene set. Use SOAPdonovo, Velvet and SPAdes short-sequence assembly software to construct images ...
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