Biomarkers for diabetes and usages thereof
A diabetes and microorganism technology, applied in the field of biomedicine, can solve the problems that the pathogenesis of type II diabetes cannot be well explained, and the type II diabetes needs to be improved.
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Embodiment 1
[0054] Example 1: Sample Collection
[0055] All 344 stool samples were collected from 344 volunteers, and stool samples were collected by Peking University Hospital, Shenzhen, China. Diabetes type II diabetes was diagnosed according to the standards issued by WHO in 1999 (Alberti, K.G.&Zimmet, P.Z. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabetic medicine: a journal of the British Diabetic Association15,539-553, doi:10.1002 / (SICI)1096-9136(199807)15:73.0.CO;2-S(1998), by reference which is incorporated herein), diagnosed type II diabetes patients as the case group, and other non-diabetic individuals as the control group (see Table 1). Type 2 diabetic patients and normal individuals need to provide frozen stool samples. Volunteers should pay attention to their diet 3 days before sampling. They should eat a light diet and not eat hig...
Embodiment 2
[0058] Example 2: DNA extraction and sequencing
[0059] 2.1 Storage of stool samples
[0060] Put the collected stool sample into the sterilized stool collection tube, and store it in the freezer immediately to freeze the stool sample. Frozen samples were sent to the storage point and stored at -80°C until use.
[0061] 2.2 DNA extraction
[0062]200 mg of frozen fecal samples were taken separately and suspended in a solution containing 250 μl of guanidine thiocyanate, 0.1 M Tris (pH 7.5) and 40 μl of 10% lauroyl sarcosine. DNA extraction method is the same as above (Manichanh, C.et al.Reduced diversity of fecal microbiota in Crohn's disease revealed by a metagenomic approach.Gut 55,205-211, doi:gut.2005.073817[pii]10.1136 / gut.2005.073817, (2006) incorporated herein by reference). DNA concentration and molecular weight were determined by Nanodrop instrument (Thermo Scientific) and agarose gel electrophoresis, respectively.
[0063] 2.3 DNA library construction and sequen...
Embodiment 3
[0067] Example 3: Identification of biomarkers
[0068] 3.1 Basic processing of sequencing data
[0069] After obtaining the sequencing data of the first phase of 145 samples, it was filtered to remove low-quality sequences containing 'N', adapter pollution sequences and host genome pollution sequences, and finally obtained 378.4Gb high-quality data. On average, high-quality data accounted for 98.1% of all data. In addition, the actual insert length of the PE library is between 313bp and 381bp.
[0070] 3.2 Updating the gene set
[0071] Using the same parameters as the MetaHIT gene set (Junjie Qin, Ruiqiang Li, Jeroen Raes, et al. (2010) A human gut microbial gene catalog established by metagenomic sequencing. Nature, 464:59-65, which is incorporated herein by reference), In the first phase, use SOAPdenovo v1.0642 and GeneMark v2.743 to perform de novo assembly and gene prediction on the sequencing sequence; then use BLAT software to compare all predicted genes, if one seq...
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