Prognosis kit for lymphoma

[0035] Example 1 Preparation of DNA

[0036] 1. Extract DNA from paraffin specimens

[0037] Place the paraffin specimen in a 1ml EP tube, use gradient xylene and alcohol for dewaxing, add 180ul ATL buffer and 20ul proteolytic enzyme, and incubate it in a 56℃ water bath overnight. After the tissue is digested, use 200ul ATL buffer and 200ul ethanol to mix well in a metal bath at 90°C for 1 hour. After centrifugation, transfer the supernatant to the adsorption column. After centrifugation at 13000rpm, centrifuge the liquid in the spin column to the collection tube and discard Collect the collection tube, put the adsorption column in a new collection tube, add 500ul AW1 buffer and centrifuge at 1200rpm for 1 min, then add AW2 500ul and centrifuge for 1 minute, put the adsorption column in a clean 1.5ml EP tube, add 100ul ATEbuffer, and place at 70℃ Centrifuge at 13000rpm for 2 minutes for 10 minutes, add 50ulPBS to 20°C and store.

[0038] 2. Extract DNA from fresh tissue

[0039] The fresh tissue was added to the lysis buffer containing proteinase K and digested at 55°C for 2 hours. The solution was cooled to room temperature, an equal volume of phenol was added, and the centrifuge tube was slowly inverted for 10 minutes to gently mix the two phases. The centrifuge tube was then placed on the rotator for 1 hour. After the two phases are separated at room temperature, transfer the water phase to another centrifuge tube. Add 0.2 times volume of 10M ammonia acetate and 2 times volume of ethanol, spin the centrifuge tube until the solution is thoroughly mixed. The precipitate was collected after centrifugation at 5000g for 5 minutes at room temperature. Use a vacuum pump to suck up the remaining ethanol and add 200ulPBS to 20°C for storage.

[0040] Example 2 Prognosis Evaluation Process

[0041] 1. Combine 1 specific sequencing primer and DNA template, and then add 200ul enzyme mixture (DNA, Polymerase, ATP sulfurylase, Luciferase, and Apyrase), and substrate mixture (APS and Luciferin).

[0042] 2. Add 1 dNTP to the reaction system. If it can just match the next base of the DNA template, under the action of DNA polymerase, the 3'end of the sequencing primer will be added, and a molecule of Pyrophosphate (PPi).

[0043] 3. Under the action of ATP sulfurylase, the generated PPi can combine with APS to form ATP; under the catalysis of luciferase, the generated ATP can form oxyluciferin with luciferin, and the specificity can be obtained through the CCD optical system The detected peak value is proportional to the number of matched bases.

[0044] 4. The remaining dNTP and a small amount of ATP in the reaction system are degraded under the action of Apyrase.

[0045] 5. Add another dNTP, repeat the 2-4 reaction, and get the corresponding value according to the obtained peak diagram.

[0046] 6. According to the four candidate methylation values, according to the formula RS=(0.812×SRPRB value)+(0.253×CELF2 value)+(1.044×HOXC5 value)-(0.163×PRR36 value). If the RS value of a patient> 1.05, it is judged that the patient is in a high-risk state (poor prognosis), and the patient is recommended to undergo a more intense treatment plan.

[0047] Specific test results such as figure 1 , figure 2 and image 3 As shown, figure 1 Where, A means LASSO-logistic test to identify recurrence-non-recurrence differential CpG sites; B means SVM-RFE algorithm to identify recurrence-non-recurrence differential CpG sites; C means LASSO-Logistic and SVM-RFE algorithm to identify candidate CpG sites Site collection; D represents heat map clustering showing recurring-non-recurring CpG sites; E represents 4 candidate CpG sites that are closely related to prognosis identified by the LASSO-Cox algorithm.

[0048] figure 2 Among them, A, B, C, and D correspond to SRPRB, CELF2, HOXC5 and PRR36 genes, respectively. Left image: The dot plot shows the distribution of 4 CpG sites in the training set, internal validation set, and external validation set; Middle image: Shows the optimal threshold of 4 CpG sites; Right image: Shown It is the relationship between the expression level of 4 CpG sites and the prognosis.

[0049] image 3 Figure A shows the distribution of CpG labels in the training set, internal validation set and external validation set; Figure B shows the optimal threshold of CpG labels; Figure C shows the relationship between the expression level of CpG labels and the prognosis Relationship.