Method and device for analyzing immunological difference between two states of individuals
A state and individual technology, applied in the field of immune difference devices, assisting in determining the individual state, and in the field of devices assisting in determining the individual state, can solve problems such as difficult liver diseases, which cannot be used as routine examinations, and complicated tissue puncture and sampling
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Embodiment 1
[0051] General methods, including:
[0052] First, sequence and identify CDR3:
[0053] Use lymphocyte separation medium to separate peripheral blood T / B lymphocytes, extract DNA (or RNA), use multiplex PCR / 5'RACE to capture CDR3, and perform high-throughput sequencing through the Hiseq2000 or Hiseq2500 or Miseq platform.
[0054] After performing quality control on the measured data, they were compared to the IMGT database (http: / / www.imgt.org / ) to determine the CDR3 sequence.
[0055] Secondly, the analysis of immune results:
[0056] The high-frequency CDR3 sequence is a highly expanded clone (highly expanded clone), and the HEC rate is defined—the highly expanded clone-rate (HEC rate) is a frequency exceeding 0.05%, preferably, a frequency not exceeding 0.5% The ratio of the number of CDR3 species to the total number of CDR3 species.
[0057] PCA analysis was performed on differentially used V subtypes, V merged subtypes (Vmerge), and / or VJ combined subtypes.
[0058] ...
Embodiment 2
[0074] Taking T lymphocytes as the research target, the optimized multiplex PCR technology is used to amplify the CDR3 region, the most diverse complementarity-determining region of the T cell receptor β chain, and the amplification primers, amplification methods, library construction and sequencing, etc. The method described in CN103205420A is carried out to obtain off-machine data, comprehensively analyze the TCR composition, evaluate the diversity of the immune system, and mine the relationship information between the immune repertoire and the occurrence and development of liver cancer, hepatitis, and rectal cancer.
[0075] The method comprises the steps of:
[0076] (1) According to the T cell receptor CDR3 sequence, design V segment and J segment primers such as CN103205420A, and reference sequence construction, including obtaining the known CDR3 sequence set from the database.
[0077] (2) Sample preparation
[0078] 1. Extract 5 mL of peripheral blood from the subject...
Embodiment 3
[0100] 17 hepatitis patient samples: including liver tissue samples and peripheral blood samples at the same time
[0101] Samples from healthy people: peripheral blood samples from 20 healthy volunteers. Normal liver tissue samples from 9 volunteers.
[0102] The PBMC isolated from peripheral blood was used as the research object for the sequencing of immune repertoire, and the content is as follows:
[0103] 1. Peripheral Blood Sampling
[0104] 1) Take 5ml of the peripheral blood sample from the patient and put it in an EDTA anticoagulant tube. Gently invert up and down 4-6 times and mix thoroughly, place at room temperature, and complete the separation of PBMC within 2 hours;
[0105] 2) Add 3 times the volume of sterile saline, and mix upside down;
[0106] 3) Take 3ml of cell layering solution in a 15ml centrifuge tube, and carefully pipette 4ml of whole blood cells diluted in step 2) along the tube wall and superimpose on the layering liquid surface, and divide into...
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