Application of product for detecting ddcfDNA in preparing product for detecting transplanted kidney injuries caused by pulmonary infection and evaluating prognosis effect

[0069] Example 1 SNP site screening

[0070] Select the dbSNP (data base SNP, https: / / www.ncbi.nlm.nih.gov / snp / ) database and the Chinese population database (stored in Shanghai Aogen Diagnostics Technology Co., Ltd.). The Chinese population database is based on 3000 cases of Chinese The list of genetic polymorphism loci constructed from the whole genome sample information, such as figure 1 As shown, there is some difference frequency information between the Chinese population database and the Hapmap database (http: / / www.hapmap.org), which represents the different genetic loci in the Chinese population, so that the Chinese population database can more accurately present the Chinese population Information about the genetic polymorphism locus.

[0071] From the above two databases, the sites with a minor allele frequency (MAF) close to 0.5 were selected, and 5754 target SNP sites as shown in Table 1 were obtained.

[0072] The SNP sites of the drug metabolism genes, such as t

[0095] Example 2 Extraction of genomic DNA and plasma cfDNA

[0096] 1. Sample collection and separation of blood cells and plasma

[0097] (1) Sample collection: Divide the blood from the donor into two parts, each about 8ml, save and transport with streck preservation tube;

[0098] (2) Plasma separation: Centrifuge a fresh whole blood sample at 1900g at 4°C for 10 minutes, separate the supernatant into a new centrifuge tube, continue to centrifuge the supernatant at 16000g, at 4°C for 10 minutes, and centrifuge the supernatant Transfer the liquid to a new centrifuge tube to obtain separated plasma, which can be stored at -20°C;

[0099] (3) Blood cell separation: the lower layer of sediment after centrifuging another fresh whole blood sample is the blood cell.

[0100] 2. Use (Laifeng Genomic DNA Extraction Kit, DK601-02) to extract the separated blood cells to obtain genomic DNA; use (QIAamp Circulating Nucleic Acid Kit, 55114) to extract the separated plasma to obtain plasma cfDNA, t

[0107] Example 3 Library construction

[0108] 1. Take 1μl of plasma cfDNA obtained in Example 2 and perform QuantiFluor TM -ST (Promega) quantification, another 1μl was used to test the quality using Agilent 2100.

[0109] 2. Using the plasma cfDNA and blood cell genomic DNA obtained in Example 2 as samples, use VAHTSUniversal DNA Library Prep Kit for Illumina V3 to prepare plasma cfDNA and genomic DNA libraries. The specific steps are as follows:

[0110] (1) End filling

[0111] a. Add 7.5μl End Repair mix to the labeled centrifuge tube;

[0112] b. Take 25μl of the quantified sample in step 2 and add the end-filling mixture shown in a to obtain a sample reaction solution with a total volume of 32.5μl, and pipette to mix evenly;

[0113] c. Run the following program on the PCR machine:

[0114] Keep at 20℃ for 15min,

[0115] Keep at 65℃ for 15min,

[0116] Let stand at 4°C.

[0117] (2) Add single-molecule labeling linker

[0118] a. Calculate the amount of reagents required according to th