CN-121975934-A - Application of reagent for detecting SNP locus in preparation of product for detecting immune rejection of organ transplant patient

CN121975934ACN 121975934 ACN121975934 ACN 121975934ACN-121975934-A

Abstract

The invention provides application of a reagent for detecting SNP loci in preparation of a product for detecting immune rejection of organ transplantation patients. The invention can realize the simultaneous monitoring of acute rejection and infection of allogeneic organ transplant patients, can quantify the genome concentration of dd-cfDNA in plasma, can realize the accurate detection of dd-cfDNA below 0.5%, and can calculate the concentration of dd-cfDNA in a sample layer, and has high sensitivity and good stability. The invention provides basis and correspondingly adjusts the treatment scheme for periodically monitoring the body condition of the transplanted patient with the allogenic organ transplantation such as kidney transplantation, heart transplantation, lung transplantation and the like, and is beneficial to realizing dynamic monitoring and timely adjustment of the treatment scheme.

Inventors

LIU SHENG
ZHENG SHANSHAN
ZHU ZHIYUAN
XU HANG
XU XINHE
LI HUAJIE
Zou Zhengbang

Assignees

广州市中治医学检验有限公司

Dates

Publication Date: 20260505
Application Date: 20260120

Claims (10)

1. The application of a reagent for detecting SNP loci in preparing a product for detecting immune rejection of an organ transplant patient is characterized in that the SNP loci are any one or more of the following 396 SNP loci: rs10910090、rs1490413、rs2890085、rs9970772、rs966366、rs1628418、rs28407457、rs612353、rs1424473、rs961378、rs927902、rs10922809、rs273875、rs3737576、rs1323723、rs1630312、rs7554936、rs7534195、rs3091244、rs560681、rs4483469、rs2274626、rs3011686、rs1329428、rs2078219、rs542321、rs12096869、rs10495407、rs12036141、rs10802869、rs876724、rs792087、rs10929913、rs4668463、rs4665591、rs7580573、rs223665、rs2278786、rs165053、rs7576036、rs13011795、rs216633、rs12620487、rs1404683、rs2597900、rs12469193、rs5010755、rs177255、rs907100、rs79099104、rs7589644、rs10204400、rs11123571、rs9287719、rs6432305、rs10195150、rs9842070、rs2565609、rs1077524、rs996000、rs377590、rs28532700、rs6777437、rs1485929、rs4364205、rs12632204、rs12636811、rs3138042、rs3773554、rs2133886、rs1509278、rs1351039、rs10433383、rs1872575、rs1401033、rs9825721、rs1558798、rs2700880、rs13091、rs9863044、rs6444724、rs2129239、rs1388866、rs10213340、rs1495505、rs13110055、rs4540055、rs528582、rs1430530、rs1006042、rs7683892、rs62332600、rs4698818、rs7677973、rs9307914、rs3906778、rs6815740、rs12500125、rs10031731、rs1810603、rs12508214、rs11727304、rs17449206、rs10155058、rs1030490、rs4702256、rs870347、rs10866586、rs40704、rs395234、rs1353061、rs4523013、rs6865840、rs28777、rs6862655、rs57909303、rs696889、rs10942817、rs330665、rs11739070、rs13182883、rs1371063、rs6897414、rs6867494、rs10515836、rs1352546、rs7722456、rs338882、rs4700908、rs12203592、rs1029047、rs1265235、rs3804474、rs13218440、rs12194361、rs4144559、rs1689239、rs1723519、rs9450001、rs313216、rs242268、rs1575100、rs6941942、rs717088、rs9373188、rs4896785、rs6905055、rs214955、rs2296510、rs556085、rs844157、rs9455722、rs752576、rs6952221、rs10281864、rs1019029、rs1178099、rs932272、rs35997004、rs10255235、rs1529590、rs10499695、rs6951369、rs7777724、rs887049、rs1922342、rs41650、rs727708、rs367311、rs6960899、rs1427585、rs321198、rs10274950、rs700275、rs579864、rs221255、rs7782393、rs13282211、rs10441668、rs1843355、rs17603389、rs17088624、rs7835202、rs10092491、rs10504269、rs16927652、rs76348100、rs10504576、rs4734233、rs720896、rs3927165、rs6984036、rs1011211、rs13273391、rs6998402、rs28722737、rs7836059、rs12155866、rs2410695、rs4741985、rs1332704、rs2383604、rs7041158、rs476578、rs4129108、rs7045684、rs2769967、rs4877811、rs11141033、rs10759835、rs10513189、rs4979453、rs204202、rs4838267、rs1463729、rs1360288、rs1053878、rs2519770、rs10776839、rs7035713、rs4880095、rs6560748、rs735155、rs2386626、rs500399、rs7905831、rs3780962、rs10763827、rs7900480、rs17287498、rs10822754、rs2574784、rs12775232、rs4918664、rs2420245、rs521230、rs3009883、rs2901257、rs985462、rs964681、rs11018033、rs2998129、rs6598055、rs12278822、rs16911971、rs645052、rs11033936、rs10742546、rs2298556、rs1893045、rs1042602、rs6591147、rs10750052、rs1024344、rs1991302、rs11216029、rs1010159、rs619208、rs7479091、rs10773937、rs10845792、rs4130389、rs10771627、rs814228、rs3847723、rs11173397、rs7972316、rs11176768、rs2307223、rs2916097、rs2238151、rs4767526、rs4366521、rs7971547、rs12816855、rs2398469、rs10773760、rs28676321、rs6490548、rs1886510、rs484312、rs2166624、rs878689、rs12583882、rs7326934、rs9563831、rs1380888、rs1394321、rs7338130、rs9300342、rs1978944、rs9577634、rs746367、rs7156806、rs7144366、rs7148442、rs950098、rs3783694、rs1958199、rs1980726、rs12434542、rs12896399、rs12895360、rs234565、rs12586773、rs873196、rs4530059、rs11633953、rs11161139、rs1800414、rs9302170、rs4924175、rs1821380、rs1528460、rs2470360、rs4619348、rs61672379、rs1372828、rs7175840、rs6598531、rs12915600、rs2342747、rs196008、rs11076452、rs1641354、rs4784376、rs1486737、rs11149894、rs4387626、rs430046、rs1382387、rs1805009、rs12603321、rs2323659、rs941454、rs4411548、rs1004357、rs8067692、rs650363、rs985492、rs2042762、rs9807731、rs1736442、rs7251928、rs539344、rs719366、rs11670999、rs385780、rs297662、rs445251、rs453573、rs2378249、rs2069945、rs1005533、rs1523537、rs310644、rs722098、rs9981337、rs2827530、rs2833736、rs987640、rs6001030、rs2024566、rs2040411、rs2704865、rs2056688、rs72611414、rs1534285、rs763056、rs1458178、rs5986751、rs993010、rs183277、rs9781645、rs1874111、rs12844754、rs7471388、rs4826248、rs6418330、rs5984589、rs1768334、rs12689741、rs5917032、rs9786184、rs9306845、rs201334828、rs17250845、rs4141886、rs16980601rs17316592、rs2032673、rs17323322 And rs17842518.
2. The use according to claim 1, wherein the reagent for detecting a SNP site comprises a primer for detecting the SNP site.
3. The use according to claim 1 or 2, wherein the reagent for detecting SNP sites detects all 396 SNP sites.
4. Use of a reagent for detecting a SNP site as set forth in claim 1 in combination with a reagent for detecting a pathogen for the preparation of a product for detecting the health condition of an organ transplant patient, characterized in that the health condition includes an immune rejection condition and an infection condition; The pathogen is any one or more of EBV, CMV, JC polyomaviruses, BK polyomaviruses, parvoviruses B19, adenoviruses B, adenoviruses C, adenoviruses E and circoviruses.
5. The use of claim 4, wherein the agent for detecting a pathogen comprises a primer for detecting the pathogen.
6. The use of claim 5, wherein the reagent for detecting a pathogen further comprises a PCR reagent.
7. A method for constructing a sequencing library for a plasma sample of an organ transplant patient is characterized in that cfDNA of the plasma sample of the organ transplant patient is extracted, then the cfDNA is amplified by using a primer for detecting the SNP locus in claim 1, end repair and A are added, connectors are connected, and the sequencing library is formed by PCR amplification.
8. The method of construction of claim 7, wherein the cfDNA is further amplified with primers that detect the pathogen of claim 4.
9. The method of claim 8, wherein the primer for detecting the pathogen has a nucleotide sequence as shown in XXX.
10. A method for assessing the health of an organ transplant patient, characterized in that a sequencing library is obtained according to the construction method of claim 8 or 9, followed by genomic sequencing, quality control and washing to remove low quality and linker sequences; Comparing the sequences of the SNP loci in the claim 1 by using BWA software, counting the reads of each SNP locus, recording all heterozygous loci in a plasma sample, and calculating according to a formula (2) to obtain MAF of each heterozygous locus, wherein the heterozygous loci are SNP loci in which wild base and mutant base exist in the plasma sample at the same time; Selecting dd-cfDNA sites by taking MAF as a threshold value for interpreting dd-cfDNA signals, and calculating according to a formula (3) to obtain an average MAF value (avg (dd-cfDNA%)) of the dd-cfDNA sites, wherein the average MAF value is used as the ratio of dd-cfDNA in the sample and is also called the relative value of dd-cfDNA of blood; formula (3) avg (dd-cfDNA%) = (a1_dd-cfDNA% + a2_dd-cfDNA% +,. The.,. Sup.+ an_dd-cfDNA%)/n, where n is the total number of dd-cfDNA sites; when avg (dd-cfDNA%) is above the organ-corresponding clinical rejection threshold, the organ transplant patient is determined to have chronic or acute immune rejection.

Description

Application of reagent for detecting SNP locus in preparation of product for detecting immune rejection of organ transplant patient Technical Field The invention relates to the technical field of detection, in particular to application of a reagent for detecting SNP loci in preparation of a product for detecting immune rejection of an organ transplant patient. Background Since the first human kidney transplantation was successfully carried out in 1954, organ transplantation has made great progress in the treatment of chronic diseases, becomes the first treatment method for many organ failure, and improves the quality of life for thousands of people. However, rejection and infection caused after organ transplantation are serious threatens to the life health of the organ recipients, so that life health monitoring and infection monitoring of transplanted organs are critical for survival of patients, and are important means for improving the long-term survival rate of the organ transplanted patients after operation. Organ transplant patients typically have an immune rejection response by continuing to take immunosuppressive drugs (such as tacrolimus), suppressing the immune system, reducing the incidence of graft rejection, but also increasing the risk of infection in patients with history of allograft transplantation. Biopsy is a "gold standard" for diagnosing whether or not there is graft rejection or infection after an organ transplant patient is operated, and is unsuitable for continuously monitoring the health status of a transplanted organ for a long period of time because it requires regular removal of organs or tissues, is invasive, and causes a certain degree of damage to the transplanted organ. In addition, diagnosis of infection requires the attending physician to identify the source of the infection correctly and to conduct a series of pathogen checks. However, infections and rejection reactions after transplantation often show similar symptoms (e.g. shortness of breath), and conventional clinical trials are often indistinguishable. Free DNA (cfDNA) is a DNA fragment present in human extracellular fluid and is currently widely used in the field of noninvasive prenatal examination and liquid biopsies of cancer. In an organ transplant patient, when the transplant is rejected by the recipient, donor cells shed from the organ, undergo apoptosis and lyse, releasing Donor-derived cfDNA (Donor-DERIVED CELL-free DNA, dd-cfDNA) into the recipient's blood. The dd-cfDNA exists in the blood plasma of various types of solid organ transplants, and a plurality of research results at present show that the dd-cfDNA is closely related to rejection-related molecules in biopsies, has important clinical reference value for diagnosing organ immune rejection and is considered to be a noninvasive real-time biomarker organ transplantation acute rejection monitoring. Circulating cfDNA concentrations in plasma are extremely low, with normal people having only hundreds to thousands of copies per milliliter (mL) of plasma, dd-cfDNA levels in organ transplant recipients are normally at extremely low levels, but with increased post-transplant immune rejection, dd-cfDNA levels rise, but due to the short half-life of cfDNA, dd-cfDNA levels drop rapidly to steady levels after rejection treatment is successful. Currently, the methods used to quantify dd-cfDNA levels in organ transplant patient recipients are mainly fluorescent quantitative PCR (qPCR), digital PCR (ddPCR), next Generation Sequencing (NGS). Early studies used y chromosome specific alleles to quantify dd-cfDNA, but were not universal. Recently there is prior art to quantify the proportion of donor specific Single Nucleotide Polymorphisms (SNPs) in acceptor cfDNA based on donor-derived PCR or NGS techniques. For example, chinese patent CN113035271a discloses a multiplex PCR primer design, primer set and detection method for detecting heterologous cfDNA, by obtaining genomic DNA of an organ transplanted recipient sample, capturing and sequencing a target region of the genomic DNA for genotyping, and simultaneously capturing and sequencing a target region of a transplanted recipient plasma cfDNA sample for analyzing the ratio of donor dd-cfDNA to total cfDNA. However, the method still has some problems in practical application, namely (1) the method is used for quantifying dd-cfDNA, the acceptor genome is required to be detected firstly for genotyping, effective SNP loci are screened based on genotyping results, dd-cfDNA duty ratio calculation is carried out, meanwhile, the genome concentration of dd-cfDNA cannot be calculated, (2) the amplicon fragment in the method is 178bp, the main peak of the cfDNA fragment is 170bp, the amplicon fragment is too long, most of cfDNA shorter than 178bp cannot be utilized, the detection sensitivity of the cfDNA is obviously affected, meanwhile, 10M reads are required for sequencing, and the sequencing cost is high, and (3) the method can