Tracking and monitoring kit for allogene transplantation as well as use method and application of tracking and monitoring kit

Abstract

The invention discloses an allogene transplantation tracking and monitoring kit which comprises a next-generation sequencing detection kit and an RT-qPCR detection kit, and the next-generation sequencing detection kit comprises a library building reagent and a recurrence monitoring site capture probe; the recurrence monitoring site capture probe comprises an HLA genotyping detection probe and a supplementary probe. According to the present invention, the site after the allogenic transplantation is screened and is combined with the HLA detection site before the transplantation, such that the additional use steps are not required, and the detection can be completed in the existing conventional detection process before the transplantation so as to avoid the complex site screening process during the monitoring after the transplantation. And the primer probe designed aiming at the InDel site is used, so that the primer probe has higher specificity and sensitivity. The gender-related genes are designed, and male recipients accepting hematopoietic stem cell transplantation from female donors can directly use sites for the gender-related genes to perform relapse monitoring, so that the experimental process is greatly simplified, the operation is simple, and the cost is low.

Description

Technical Field

[0001] The present invention relates to the field of testing or analyzing materials by measuring the chemical or physical properties of the materials, and particularly relates to an allogeneic transplantation tracking monitoring kit based on NGS and fluorescence PCR, a using method and an application thereof. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation is an effective means for treating various hematological diseases such as leukemia, myelodysplastic syndrome, multiple myeloma, non-Hodgkin lymphoma, etc. However, only a small number of patients have clear and suitable fusion genes or gene mutations for relapse monitoring after allogeneic hematopoietic stem cell transplantation. The existing gene tracking and monitoring methods after transplantation are relatively complex, requiring detection of all loci, or having a long detection period and high detection cost, and cannot meet the need for early detection of relapse and carrying out targeted treatment. Therefore, it is crucial to develop a gene tracking and monitoring method for recipients before and after transplantation to enable timely relapse monitoring.

[0003] Chinese invention patent CN110423818A discloses a primer, a probe, a kit and a method for HLA-LOSS detection and individual identification. Based on HLA loci with a high mismatch rate in the Asian population in the latest IMGT database, and aiming at HLA loci with a high mismatch rate, a specific primer and probe combination is further designed, which has the advantages of good specificity, high sensitivity and simplicity and rapidity. However, it is only a kit based on primers and probes, with low sensitivity. Chinese invention patent CN106029903B discloses a method and a probe for identifying alleles of a gene, a method for genotyping highly polymorphic alleles (such as HLA alleles) using high-throughput sequencing technology, which is suitable for identifying alleles in highly polymorphic genes by targeting capture probes to non-coding sequences. However, the length of the probe is long and the preparation method is cumbersome. Summary of the Invention

[0004] In order to develop a method that can perform gene tracking and monitoring for recipients before and after transplantation and enable timely relapse monitoring, multiple InDel loci are screened, and a HLA high-resolution typing detection supplementary probe compatible with RNA probe type and for high-throughput HLA high-resolution typing detection is designed and synthesized. It can perform typing judgment on candidate InDel loci of donors and recipients while performing HLA gene typing detection by NGS method before transplantation of donors and recipients, and determine the loci that can be used for relapse monitoring, so that there is no need to perform screening after transplantation; on the other hand, we design and screen specific primers and probes, so that patients after transplantation can perform experiments on 1 differential locus by RT-qPCR method to achieve relapse monitoring.

[0005] The first aspect of the present invention provides an allogeneic transplantation tracking and monitoring kit, which includes a next-generation sequencing detection kit and an RT-qPCR detection kit. The next-generation sequencing detection kit at least includes a library construction reagent and a relapse monitoring locus capture probe.

[0006] The next-generation sequencing detection kit (HLA high-resolution detection) is used for screening and tracking monitoring loci before allogeneic transplantation; the RT-qPCR detection kit is used for monitoring the chimerism rate after allogeneic transplantation.

[0007] As a preferred embodiment, the library construction reagent at least includes a fragment and end repair solution, a fragment and end repair enzyme, a ligase, a pre-PCR amplification reaction solution, a unique dual-tag primer, a purification magnetic bead, a capture magnetic bead, a post-PCR amplification reaction solution, and a post-PCR amplification primer (from Aegtek Biotechnology Co., Ltd.).

[0008] As a preferred embodiment, the library construction reagent includes a fragment and end repair solution, a fragment and end repair enzyme, a ligation buffer, a ligase, a universal adaptor, a pre-PCR amplification reaction solution, a unique dual-tag primer, a purification magnetic bead, a hybridization buffer, a blocking solution 1, a blocking solution 2, a probe protection solution, a capture magnetic bead, a post-PCR amplification reaction solution, a post-PCR amplification primer, a binding buffer, a washing buffer 1, and a washing buffer 2 (from Aegtek Biotechnology Co., Ltd.).

[0009] As a preferred embodiment, the relapse monitoring locus capture probe includes an HLA genotyping detection probe and an HLA high-resolution genotyping detection supplementary probe.

[0010] As a preferred embodiment, the RT-qPCR detection kit includes a locus reaction solution, a PCR MIX, and a negative control.

[0011] As a preferred embodiment, the locus gene frequency in the locus reaction solution is 0.25 - 0.75.

[0012] As a preferred embodiment, the number of inserted or deleted bases at the locus in the locus reaction solution > 3.

[0013] Common chromosomal variations in leukemia include: BCR-ABL t(9;22)(q13;q11), PML-RARA t(15;17)(q22;q21), RUNX1-RUNX1T1 t(8;21)(q22;q22), CBFB-MYH11 inv(16)(p13.1q22), MLL-AF9 t(9;11), ETV6-RUNX1 t(12;21), KMT2A rearrangement t(v;11q23). When designing and selecting relapse tracking and monitoring sites, the inventors avoided selecting InDel sites near the chromosomes or regions where common fusion genes are located. And according to the East Asian distribution frequency in the genome1000 database, sites with gene frequencies between 0.25 and 0.75 were selected as relapse tracking and monitoring sites to ensure the discrimination of the sites.

[0014] Considering that one site may have multiple insertion and deletion base sequences, it is necessary to design multiple reaction tubes, resulting in an excessive number of reaction tubes in the kit. Therefore, the inventors selected sites with only one type of insertion or deletion sequence type and achieved precise positioning detection by designing two reaction tubes. At the same time, the inventors selected relapse tracking and monitoring Indel sites with the number of inserted and deleted bases > 3, which can make the specificity of primers and probes in the RT-qPCR reaction solution better.

[0015] As a preferred embodiment, the sites in the site reaction solution include at least one of rs2307700, rs2308010, rs5787309, rs3067397, rs66595817, rs3834231, rs33971783, rs34855933, rs10558392, rs3042783, rs71644113, rs3038530, rs67426579 or the human sex-determining gene SRY site.

[0016] The information of the sites is shown in Table 1 below.

[0017] Table 1

[0018] rs number Chromosome position Allele-1 Allele-2 Allele-1 frequency Allele-2 frequency rs2307700 chr22:26394936 TCA TCACTCA 0.2599 0.7401 rs2308010 chr1:91029816 AG AGTCAG 0.5764 0.4236 rs5787309 chr10:98769092 TTATTTTA TTA 0.5407 0.4593 rs3067397 chr1:61320635 TATCTTA TA 0.3105 0.6895 rs66595817 chr5:114472240 CTTTC - 0.7004 0.2996 rs3834231 chr4:8376859 TAGG - 0.6113 0.3887 rs33971783 chr19:15268500 TTTG - 0.3829 0.6171 rs34855933 chr1:241888410 GTCT - 0.6002 0.3998 rs10558392 chr2:44508578 AAAC - 0.6766 0.3234 rs3042783 chr2:221296039 AA AACTCAA 0.5704 0.4296 rs71644113 chr7:134142604 AAGA - 0.6796 0.3204 rs3038530 chr13:111892610 GATT - 0.5258 0.4742 rs67426579 chr7:77203403 TGG - 0.4583 0.5417

[0019] "-" means that the site does not have the previous sequence, which is based on the ncbi database identification.

[0020] The human sex-determining gene SRY site is located on the Y chromosome of males and can be directly used to detect the situation of female donors and male recipients.

[0021] According to the cumulative individual recognition rate calculation method in the "Technical Specification for Individual Identification", the cumulative individual recognition rate of the above 13 loci is greater than 0.9999. When performing follow-up monitoring, it is more accurate to detect the alleles that the recipient has but the donor does not have. Based on this calculation, the probability that there are alleles available for monitoring between the donor and the recipient is greater than 0.99.

[0022] As a preferred embodiment, the primer and probe sequences of each locus of the RT-qPCR detection kit after allogeneic transplantation are shown in Table 2 below:

[0023] Table 2

[0024]

[0025]

[0026] The second aspect of the present invention provides a method for using an allogeneic transplantation follow-up monitoring kit, including the following steps:

[0027] S1 Extract the DNA of the recipient before transplantation and the donor before transplantation;

[0028] S2 Use the DNA extracted in step S1 and perform PCR amplification with a next-generation sequencing detection kit. During the PCR amplification process, introduce HLA gene typing detection probes and HLA high-resolution typing detection supplementary probes. After completion, obtain a DNA final library;

[0029] S3 Sequence and compare the DNA final library, and use the loci that the recipient has but the donor does not have as subsequent recurrence monitoring loci;

[0030] S4 Extract the DNA of the recipient after transplantation or the cfDNA of the recipient after transplantation;

[0031] S5 Use the DNA or cfDNA extracted in step S4 and perform PCR amplification with an RT-qPCR detection kit;

[0032] S6 Detect, interpret the results, and analyze the results.

[0033] As a preferred embodiment, the specific steps of step S2 are:

[0034] 2.1 Take out the fragment and end repair buffer and the fragment and end repair enzyme and melt them on an ice box. After the reagents are melted, on the ice box, according to 100 ng of DNA, 4 μL of fragment and end repair buffer, 6 μL of fragment and end repair enzyme, and use nuclease-free water to make up the reaction volume to 50 μL. Use a pipette to blow and mix evenly, and centrifuge. Set the thermal cover of the PCR instrument to 105 °C, set the PCR program to 4 °C for 1 min; 37 °C for 18 min; 65 °C for 30 min; keep at 4 °C. Place the reaction system on the PCR instrument for reaction;

[0035] 2.2 Take out the universal adapter, ligase, and ligation buffer and melt them on an ice box. After the PCR program in step 2.1 is completed, take out the reaction product, add 5 μL of the universal adapter, 14 μL of the ligation buffer, and 7 μL of the ligase. Mix well by pipetting and centrifuge. Set the hot lid of the PCR instrument to Off, set the PCR program to 20 °C for 15 min; 4 °C for incubation; Place the reaction system on the PCR instrument for reaction;

[0036] 2.3 Place the purification magnetic beads at room temperature 30 min in advance. After the PCR program in step 2.2 is completed, transfer the reaction product to a new 1.5 mL centrifuge tube and add 60 μL of the purification magnetic beads; Let it stand at room temperature for 5 min, place the centrifuge tube on the magnetic rack for 5 min until the solution becomes clear; Keep the centrifuge tube on the magnetic rack, remove the supernatant, add 180 μL of 80 vol% ethanol, let it stand for 30 s, remove the supernatant and repeat once, let it stand at room temperature for 5 min to completely volatilize the residual ethanol, add 17 μL of nuclease-free water (from Nanjing Novoprotein Scientific Co., Ltd.), remove the centrifuge tube from the magnetic rack, gently pipette to resuspend the magnetic beads, let it stand at room temperature for 2 min, place the centrifuge tube on the magnetic rack for 2 min until the solution becomes clear, and use a pipette to aspirate 15 μL of the supernatant and transfer it to a new PCR tube;

[0037] 2.4 Place the pre-PCR amplification reaction solution and the unique dual-tag primer on an ice box to melt. Prepare the reaction system according to 15 μL of the purified product obtained in step 2.3, 25 μL of the pre-PCR amplification reaction solution, and 10 μL of the unique dual-tag primer. Set the PCR hot lid to 105 °C, set the PCR program to 98 °C for 45 s; (98 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s) × 8 cycles, 72 °C for 1 min, 4 °C for incubation. Place the reaction system on the PCR instrument for reaction. After the amplification program is completed, proceed to the next step immediately;

[0038] 2.5 Place the purification magnetic beads at room temperature 30 min in advance. After the PCR program in step 2.4 is completed, transfer the reaction product to a new 1.5 mL centrifuge tube and add 50 μL of the purification magnetic beads; Let it stand at room temperature for 5 min, place the centrifuge tube on the magnetic rack for 5 min until the solution becomes clear; Keep the centrifuge tube on the magnetic rack, remove the supernatant, add 180 μL of 80 vol% ethanol, let it stand for 30 s, remove the supernatant and repeat once, let it stand at room temperature for 5 min to completely volatilize the residual ethanol, add 22 μL of nuclease-free water, remove the centrifuge tube from the magnetic rack, gently pipette to resuspend the magnetic beads, let it stand at room temperature for 2 min, place the centrifuge tube on the magnetic rack for 2 min until the solution becomes clear, use a pipette to aspirate 20 μL of the supernatant and transfer it to a new PCR tube, make a mark, and proceed with the subsequent experimental steps or store the pre-library at -20 ± 4 °C;

[0039] 2.6 Four pre-libraries constructed using 4 different unique dual-tag primers were mixed and hybridized, with 300 ng of each pre-library. The mixed centrifuge tube was placed in a centrifugal concentrator. The lid of the PCR tube was opened, the centrifugal concentrator was started, and concentration was carried out until it was in a dry state. Take out Blocking Solution 1, Blocking Solution 2, Probe Protection Solution, HLA Genotyping Detection Probe, and HLA High-Resolution Genotyping Detection Supplementary Probe and melt them on an ice box. Add 13 μL of Hybridization Buffer, 5 μL of Blocking Solution 1, 2 μL of Blocking Solution 2, 5 μL of Probe Protection Solution, 1 - 2 μL of HLA Genotyping Detection Probe, and 0.2 - 1 μL of HLA High-Resolution Genotyping Detection Supplementary Probe to the concentrated centrifuge tube. Use nuclease-free water to make up the volume of the reaction system to 30 μL. Gently pipette and mix well, and briefly centrifuge. Set the thermal lid of the PCR instrument to 105 °C, set the PCR program to 80 °C for 5 min, 50 °C for 16 h, and hold at 50 °C. Place the reaction system on the PCR instrument for reaction;

[0040] 2.7 Place the capture beads at room temperature 30 min in advance. Take 50 μL of capture beads into a new centrifuge tube, place it on a magnetic stand for 1 min until the solution becomes clear, remove the supernatant. Take the centrifuge tube off the magnetic stand, add 180 μL of Binding Buffer, gently pipette to resuspend the beads, place it on the magnetic stand for 1 min until the solution becomes clear, remove the supernatant. Repeat the bead washing once. Take the centrifuge tube off the magnetic stand, add 180 μL of Binding Buffer, gently pipette to resuspend the beads, and set aside;

[0041] 2.8 Keep the reaction system on the PCR instrument, add 180 μL of capture beads, gently pipette and mix well with a pipette, place it on a thermostatic shaking mixer, and bind at room temperature for 30 min. Place the centrifuge tube on the magnetic stand for 2 min until the solution becomes clear, remove the supernatant. Add 150 μL of Wash Buffer 1, gently pipette and mix well with a pipette, place it on a thermostatic shaking mixer, and bind at room temperature for 15 min. Place the centrifuge tube on the magnetic stand for 2 min until the solution becomes clear, remove the supernatant. Add 150 μL of Wash Buffer 2 preheated to 50 °C, gently pipette and mix well with a pipette, place it on a thermostatic shaking mixer, and incubate at 50 °C for 10 min. Place the centrifuge tube on the magnetic stand for 2 min until the solution becomes clear, remove the supernatant; Repeat the washing of the beads twice with Wash Buffer 2 preheated to 50 °C, for a total of 3 washes; Keep the centrifuge tube on the magnetic stand, remove the supernatant, add 180 μL of 80 vol% ethanol, let it stand for 30 s, remove the supernatant; Let it stand at room temperature for 5 min to allow the residual ethanol to completely volatilize; Add 24 μL of nuclease-free water, take the centrifuge tube off the magnetic stand, and gently pipette to resuspend the beads;

[0042] 2.9 After taking out the post-PCR amplification reaction solution and post-PCR amplification primers, thaw them on an ice box. Add 25 μL of the post-PCR amplification reaction solution and 1 μL of the post-PCR amplification primers to the final product (sample containing magnetic beads) in step 2.8. Set the PCR hot lid to 105 °C, and set the PCR program as 95 °C for 1 min, (98 °C for 20 s, 60 °C for 30 s, 72 °C for 30 s) × 12 - 14 cycles, 72 °C for 5 min, and hold at 4 °C.

[0043] 2.10 After the PCR program in step 2.9 is completed, add 55 μL of purified magnetic beads, gently pipette and mix well, let stand at room temperature for 5 min, place the centrifuge tube on a magnetic rack for 3 min until the solution becomes clear. Keep the centrifuge tube on the magnetic rack, remove the supernatant, add 180 μL of 80 vol% ethanol, let stand for 30 s, remove the supernatant and repeat once. Let stand at room temperature for 5 min to completely volatilize the residual ethanol. Add 25 μL of nuclease-free water, remove the centrifuge tube from the magnetic rack, gently pipette to resuspend the magnetic beads, let stand at room temperature for 2 min, place the centrifuge tube on the magnetic rack for 2 min until the solution becomes clear, and pipette 23 μL of the supernatant into a new centrifuge tube to obtain the DNA final library.

[0044] As a preferred embodiment, the specific steps of step S5 are as follows:

[0045] 5.1 Take out the reaction solution and PCR MIX corresponding to the chimerism tracking monitoring site after recipient hematopoietic stem cell transplantation from the kit, thaw at room temperature and mix well by oscillation, then centrifuge at 2000 rpm for 10 s. The PCR premix prepared for the reaction system is as follows: 7 μL of the site reaction solution and 13 μL of PCR MIX (the number of reaction tubes is the sum of the number of samples and the negative control). Aliquot the prepared PCR premix into centrifuge tubes at 20 μL per tube.

[0046] 5.2 Loading samples: Take 5 μL of the negative control and DNA samples respectively, add them to the centrifuge tubes containing the PCR premix, tighten the tube caps, and transfer them to the detection area.

[0047] As a preferred embodiment, the PCR amplification program of step S5 is 42 °C for 5 min; 94 °C for 3 min; then cycle 40 - 50 times at 94 °C for 15 s and 60 °C for 60 s, and collect the fluorescence signal at the end of the last cycle. The detection channel is FAM or VIC.

[0048] As a preferred embodiment, when the sample is DNA, the PCR amplification program of step S5 is 42 °C for 5 min; 94 °C for 3 min; then cycle 40 times at 94 °C for 15 s and 60 °C for 60 s. The reaction system is 25 μL, and collect the fluorescence signal at the end of the last cycle. The detection channel is FAM or VIC.

[0049] As a preferred embodiment, when the sample is cfDNA, the PCR amplification program in step S5 is 42°C for 5 min; 94°C for 3 min; then 94°C for 15 s and 60°C for 60 s for 50 cycles. The reaction system is 25 μL. Fluorescence signals are collected at the end of the last cycle, and the detection channels are FAM and VIC.

[0050] As a preferred embodiment, the result interpretation in step S6 is specifically as follows:

[0051] (1) Determination of the baseline: The software default sets the average fluorescence signal of 3 - 15 cycles as the baseline. In the experiment, select the section with smaller curve fluctuations and greater stability as the baseline. The starting point should avoid the signal increase caused by high temperature in the first few cycles and be set at the place where the signal has dropped to the background level and can maintain stability. The end point should avoid covering the place where the signal has started to increase significantly. According to the different trends of the experimental curve, the stop value is selected at the position of 2 - 3 Ct values before the first obvious inflection point of the entire amplification curve; the start value is selected with an interval of more than 8 cycles between the starting point and the end point to better meet the mathematical requirements for statistically calculating the baseline standard deviation.

[0052] (2) Determination of the threshold: In the case of no amplification in the negative control, the threshold is set at the highest point of the sample without an amplification curve, that is, higher than the highest point of the non - amplification growth curve, and on the principle that no detection is found in the negative control, the starting threshold is determined.

[0053] As a preferred embodiment, in the result analysis of step S6, the validity determination is as follows: The CT value of the negative control must be ≥ 38 or show "Undet"; the DNA sample of the patient before transplantation is used as a positive control, and its CT value should be ≤ 32.0 for the data to be valid.

[0054] As a preferred embodiment, in the qualitative determination in the result analysis of step S6, it is determined according to the difference (ΔCT) between the CT value of the target gene and the CT value of the reference gene. When the CT value of the reference gene in the specimen is ≤ 28, if ΔCT > 12, it is determined that the target gene of the recipient before transplantation does not exist in the sample after allogeneic transplantation of the recipient; if ΔCT ≤ 12, it is determined that the target gene of the recipient before transplantation exists in the sample after allogeneic transplantation of the recipient, and then quantitative calculation is performed.

[0055] As a preferred embodiment, the specific calculation of the chimerism rate of the DNA sample in the result analysis of step S6 is as follows: Denote the CT values of the target gene and the reference gene of the recipient's sample before transplantation as CT (target gene before transplantation) and CT (reference gene before transplantation) respectively; denote the CT values of the target gene and the reference gene of the recipient's sample after transplantation as CT (target gene after transplantation) and CT (reference gene after transplantation) respectively.

[0056] ΔΔC T = [C T (Post-transplantation monitoring site) - C T (Post-transplantation reference gene)] - [C T (Pre-transplantation monitoring site)

[0057] -C T (Pre-transplantation reference gene)]

[0058]

[0059] If multiple sites are detected: (N is the number of detected sites)

[0060] (3) Quantitative calculation of cfDNA sample: Calculate cfDNA equivalent and donor-derived cfDNA ratio:

[0061]

[0062] ΔC T = C T (Monitoring site) - C T (Reference gene)

[0063] The third aspect of the present invention provides an application of an allogeneic transplantation tracking and monitoring kit, which is applied to hematopoietic stem cell transplantation or solid organ transplantation.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] (1) The allogeneic transplantation tracking and monitoring kit of the present invention combines the screening of sites after allogeneic transplantation with the HLA detection sites before transplantation, without the need to add additional usage steps, and can be completed during the existing routine pre-transplantation detection process, thus avoiding the complex site screening process during post-transplantation monitoring.

[0066] (2) The allogeneic transplantation tracking and monitoring kit of the present invention uses primers and probes designed for InDel, a multi-base mutation site, which has higher specificity and sensitivity compared to primers and probes for single-base mutation sites.

[0067] (3) The allogeneic transplantation tracking and monitoring kit of the present invention excludes InDel sites located on the chromosomes or regions where common leukemia fusion genes are located, reducing the interference affected by leukemia fusion genes;

[0068] (4) The allogeneic transplantation tracking and monitoring kit of the present invention designs for gender-related genes, and for male recipients who receive hematopoietic stem cell transplantation from female donors, the sites for gender-related genes can be directly used for relapse monitoring.

[0069] (5) The allogeneic transplantation tracking and monitoring reagent kit of the present invention can obtain the corresponding locus information while performing HLA high-resolution genotyping detection before transplantation, without the need to detect each locus of the sample after transplantation. When performing RT-qPCR detection, the present invention does not require nested amplification, and only one-step fluorescence PCR is needed to detect the chimerism rate of the sample, greatly simplifying the experimental process, with simple operation, short experimental period, and low cost.

[0070] (6) The allogeneic transplantation tracking and monitoring reagent kit of the present invention has good specificity, can detect ddcfDNA, monitor the cell state of solid organs, has a low detection limit, is convenient for early detection of recurrence, and has good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 is the detection flow chart of Example 1;

[0072] Figure 2 is the amplification curve graph for verifying the specificity of the reaction solution at the rs3067397 locus in Example 1; top: rs3067397 allele-1 reaction solution; bottom: rs3067397 allele-2 reaction solution;

[0073] Figure 3 is the amplification curve graph for verifying the accuracy of rs3067397 in Example 1; top: rs3067397 allele-1 reaction solution; bottom: rs3067397 allele-2 reaction solution;

[0074] Figure 4 is the amplification curve graph for verifying the repeatability of rs3067397 in Example 1; top: rs3067397 allele-1 reaction solution; bottom: rs3067397 allele-2 reaction solution;

[0075] Figure 5 is the PCR amplification curve graph for detecting the cfDNA sample in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0076] Example 1

[0077] An allogeneic transplantation tracking and monitoring reagent kit includes a next-generation sequencing detection kit and an RT-qPCR detection kit. The next-generation sequencing detection kit includes at least a library construction reagent and a recurrence monitoring locus capture probe.

[0078] The library construction reagents include fragment and end repair solution, fragment and end repair enzyme, ligation buffer, ligase, universal adapter, pre-PCR amplification reaction solution, unique dual-tag primer, purification magnetic beads, hybridization buffer, blocking solution 1, blocking solution 2, probe protection solution, capture magnetic beads, post-PCR amplification reaction solution, post-PCR amplification primer, binding buffer, washing buffer 1, and washing buffer 2 (from Aegitek Biotechnology Co., Ltd.).

[0079] The relapse monitoring site capture probes include HLA genotyping detection probes and HLA high-resolution genotyping detection supplementary probes.

[0080] The site in the site reaction solution is rs3067397, and the site reaction solution includes rs3067397 allele-1 reaction solution and rs3067397 allele-2 reaction solution. The specific information of the site is shown in Table 1.

[0081] The primer and probe sequences of the rs3067397 site in the RT-qPCR detection kit are shown in Table 2.

[0082] A method for using an allogeneic transplantation tracking and monitoring kit includes the following steps:

[0083] S1 Extract the DNA of the recipient before transplantation and the donor before transplantation;

[0084] S2 Perform PCR amplification on the DNA extracted in step S1 using a next-generation sequencing detection kit, introduce HLA genotyping detection probes and HLA high-resolution genotyping detection supplementary probes during the PCR amplification process, and obtain the final DNA library after completion;

[0085] S3 Sequence and compare the final DNA library, and use the sites that the recipient has but the donor does not have as the subsequent relapse monitoring sites;

[0086] S4 Extract the DNA of the recipient after transplantation;

[0087] S5 Perform PCR amplification on the DNA extracted in step S4 using an RT-qPCR detection kit;

[0088] S6 Detect, interpret the results, and analyze the results.

[0089] The specific steps of step S2 are as follows:

[0090] 2.1 Take out the fragment and end repair buffer, and the fragment and end repair enzyme and melt them on an ice box. After the reagents are melted, on the ice box, add 100 ng of DNA, 4 μL of the fragment and end repair buffer, 6 μL of the fragment and end repair enzyme, and make up the reaction volume to 50 μL with nuclease-free water. Mix well by pipetting and centrifuge. Set the hot lid of the PCR instrument to 105 °C, and set the PCR program as follows: 4 °C for 1 min; 37 °C for 18 min; 65 °C for 30 min; keep at 4 °C. Place the reaction system on the PCR instrument for reaction.

[0091] 2.2 Take out the universal adapter, ligase, and ligation buffer and melt them on an ice box. After the PCR program in step 2.1 is completed, take out the reaction product, add 5 μL of the universal adapter, 14 μL of the ligation buffer, and 7 μL of the ligase. Mix well by pipetting and centrifuge. Set the hot lid of the PCR instrument to Off, and set the PCR program as follows: 20 °C for 15 min; keep at 4 °C. Place the reaction system on the PCR instrument for reaction.

[0092] 2.3 Place the purification magnetic beads at room temperature 30 min in advance. After the PCR program in step 2.2 is completed, transfer the reaction product to a new 1.5 mL centrifuge tube, and add 60 μL of the purification magnetic beads; let stand at room temperature for 5 min, place the centrifuge tube on a magnetic rack for 5 min until the solution becomes clear; keep the centrifuge tube on the magnetic rack, remove the supernatant, add 180 μL of 80 vol% ethanol, let stand for 30 s, remove the supernatant and repeat once. Let stand at room temperature for 5 min to completely volatilize the residual ethanol. Add 17 μL of nuclease-free water (from Nanjing Novoprotein Scientific Co., Ltd.), remove the centrifuge tube from the magnetic rack, gently pipette to resuspend the magnetic beads, let stand at room temperature for 2 min, place the centrifuge tube on the magnetic rack for 2 min until the solution becomes clear, and use a pipette to transfer 15 μL of the supernatant to a new PCR tube.

[0093] 2.4 Take out the pre-PCR amplification reaction solution and the unique dual-tag primer and melt them on an ice box. Prepare the reaction system according to 15 μL of the purified product obtained in step 2.3, 25 μL of the pre-PCR amplification reaction solution, and 10 μL of the unique dual-tag primer. Set the PCR hot lid to 105 °C, and set the PCR program as follows: 98 °C for 45 s; (98 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s) × 8 cycles, 72 °C for 1 min, keep at 4 °C. Place the reaction system on the PCR instrument for reaction. Immediately proceed to the next step after the amplification program is completed.

[0094] 2.5 Place the purified magnetic beads at room temperature 30 min in advance. After the PCR program in step 2.4 is completed, transfer the reaction product to a new 1.5 mL centrifuge tube, and add 50 μL of purified magnetic beads; let it stand at room temperature for 5 min, place the centrifuge tube on a magnetic stand for 5 min until the solution becomes clear; keep the centrifuge tube on the magnetic stand, remove the supernatant, add 180 μL of 80 vol% ethanol, let it stand for 30 s, remove the supernatant and repeat once, let it stand at room temperature for 5 min to completely volatilize the residual ethanol, add 22 μL of nuclease-free water, remove the centrifuge tube from the magnetic stand, gently pipette to resuspend the magnetic beads, let it stand at room temperature for 2 min, place the centrifuge tube on the magnetic stand for 2 min until the solution becomes clear, pipette 20 μL of the supernatant and transfer it to a new PCR tube, make a mark, and proceed with the subsequent experimental steps or store the pre-library at -20 ± 4 °C;

[0095] 2.6 Mix and hybridize four pre-libraries constructed with different unique dual-tag primers, 300 ng for each pre-library. Place the mixed centrifuge tube into a centrifugal concentrator, open the lid of the PCR tube, start the centrifugal concentrator, and concentrate until it is in a dry state. Take out Blocking Solution 1, Blocking Solution 2, Probe Protection Solution, HLA Genotyping Detection Probe, and HLA High-Resolution Typing Detection HLA High-Resolution Typing Detection Supplementary Probe and melt them on an ice box. Add 13 μL of hybridization buffer, 5 μL of Blocking Solution 1, 2 μL of Blocking Solution 2, 5 μL of Probe Protection Solution, 2 μL of HLA Genotyping Detection Probe, and 1 μL of HLA High-Resolution Typing Detection HLA High-Resolution Typing Detection Supplementary Probe to the concentrated centrifuge tube, and supplement the reaction system volume to 30 μL with nuclease-free water. Gently pipette to mix well and centrifuge briefly. Set the thermal cover of the PCR instrument to 105 °C, set the PCR program to 80 °C for 5 min, 50 °C for 16 h, and keep it at 50 °C for incubation. Place the reaction system on the PCR instrument for reaction;

[0096] 2.7 Place the capture magnetic beads at room temperature 30 min in advance. Take 50 μL of capture magnetic beads into a new centrifuge tube, place it on a magnetic stand for 1 min until the solution becomes clear, remove the supernatant, take the centrifuge tube off the magnetic stand, add 180 μL of binding buffer, gently pipette to resuspend the magnetic beads, place it on the magnetic stand for 1 min until the solution becomes clear, remove the supernatant, repeat washing the magnetic beads once, take the centrifuge tube off the magnetic stand, add 180 μL of binding buffer, gently pipette to resuspend the magnetic beads, and set aside;

[0097] 2.8 Keep the reaction system on the PCR instrument, add 180 μL of capture magnetic beads, gently pipette and mix well, place it on a constant temperature shaking mixer, bind at room temperature for 30 min, place the centrifuge tube on the magnetic rack for 2 min until the solution is clear, remove the supernatant, add 150 μL of washing buffer 1, gently pipette and mix well, place it on a constant temperature shaking mixer, bind at room temperature for 15 min; place the centrifuge tube on the magnetic rack for 2 min until the solution is clear, remove the supernatant, add 150 μL of washing buffer 2 preheated to 50 °C, gently pipette and mix well, place it on a constant temperature shaking mixer, incubate at 50 °C for 10 min. Place the centrifuge tube on the magnetic rack for 2 min until the solution is clear, remove the supernatant; repeat the washing of the magnetic beads twice with washing buffer 2 preheated to 50 °C, for a total of 3 washes; keep the centrifuge tube on the magnetic rack, remove the supernatant, add 180 μL of 80 vol% ethanol, let stand for 30 s, remove the supernatant; let stand at room temperature for 5 min to completely volatilize the residual ethanol; add 24 μL of nuclease-free water, remove the centrifuge tube from the magnetic rack, gently pipette to resuspend the magnetic beads;

[0098] 2.9 Take out the post-PCR amplification reaction solution and post-PCR amplification primers and melt them on an ice box. Add 25 μL of the post-PCR amplification reaction solution and 1 μL of the post-PCR amplification primer to the end product (sample containing magnetic beads) in step 2.8; set the PCR hot lid to 105 °C, set the PCR program as 95 °C, 1 min, (98 °C, 20 s, 60 °C, 30 s, 72 °C, 30 s) × 13 cycles, 72 °C, 5 min, hold at 4 °C;

[0099] 2.10 After the PCR program in step 2.9 is completed, add 55 μL of purification magnetic beads, gently pipette and mix well, let stand at room temperature for 5 min, place the centrifuge tube on the magnetic rack for 3 min until the solution is clear, keep the centrifuge tube on the magnetic rack, remove the supernatant, add 180 μL of 80 vol% ethanol, let stand for 30 s, remove the supernatant and repeat once, let stand at room temperature for 5 min to completely volatilize the residual ethanol, add 25 μL of nuclease-free water, remove the centrifuge tube from the magnetic rack, gently pipette to resuspend the magnetic beads, let stand at room temperature for 2 min, place the centrifuge tube on the magnetic rack for 2 min until the solution is clear, use a pipette to aspirate 23 μL of the supernatant and transfer it to a new centrifuge tube to obtain the DNA final library.

[0100] The specific steps of step S5 are as follows:

[0101] 5.1 Take out the reaction solution corresponding to the chimerism tracking and monitoring sites after hematopoietic stem cell transplantation of the recipient and PCR MIX from the kit. After melting at room temperature and mixing by oscillation, centrifuge at 2000 rpm for 10 s. The PCR premix prepared for the reaction system is as follows: 7 μL of the site reaction solution and 13 μL of PCR MIX (the number of reaction solution tubes is the sum of the number of samples and the negative control). Divide the above-prepared PCR premix into centrifuge tubes at a volume of 20 μL per tube;

[0102] 5.2 Sample addition: Take 5 μL of the negative control and DNA sample respectively, add them to the centrifuge tubes containing the PCR premix, tighten the tube caps, and transfer them to the detection area.

[0103] The sample is DNA. The PCR amplification program in step S5 is 42 °C for 5 min; 94 °C for 3 min; then cycle 40 times at 94 °C for 15 s and 60 °C for 60 s. The reaction system is 25 μL. Collect the fluorescence signal at the end of the last cycle, and the detection channel is FAM.

[0104] The result interpretation in step S6 is specifically as follows:

[0105] (1) Determination of the baseline: The software default sets the average fluorescence signal of 15 cycles as the baseline. In the experiment, select the section with less curve fluctuation and more stability as the baseline. The starting point should avoid the signal increase caused by high temperature in the first few cycles, and be set at the place where the signal has dropped to the background height and can maintain stability. The end point should avoid covering the place where the signal has started to increase significantly. According to the different trends of the experimental curve, the stop value is selected at the position of 3 Ct values before the first obvious inflection point of all amplification curves; the start value is selected with an interval of more than 8 cycles between the starting point and the end point to better meet the mathematical requirements for statistically calculating the baseline standard deviation.

[0106] (2) Determination of the threshold: In the case of no amplification of the negative control, the threshold is set at the highest point of the sample without an amplification curve, that is, higher than the highest point of the non-amplifying growth curve, and the starting threshold is determined on the principle that the negative control is not detected.

[0107] Example 2

[0108] An allogeneic transplantation tracking and monitoring kit, the specific implementation method is the same as that in Example 1, the difference is that the site reaction solution is the rs2307700 allele-1 reaction solution, and the cfDNA sample of the patient after transplantation is used.

[0109] Extract the cfDNA of the recipient after transplantation in step S4.

[0110] The sample is cfDNA. The PCR amplification program in step S5 is 42°C for 5 min; 94°C for 3 min; then 94°C for 15 s and 60°C for 60 s for 50 cycles. The reaction system is 25 μL. Fluorescence signals are collected at the end of the last cycle, and the detection channels are FAM and VIC.

[0111] Performance test

[0112] 1. Verification of the effect of supplementary probes for HLA high-resolution typing detection: According to the implementation steps of Example 1, after sequencing, the HLA high-resolution detection results are consistent with the reference results. The HLA high-resolution detection results and the reference results are shown in Table 3, indicating that after adding the supplementary probes for HLA high-resolution typing detection, the genotypes of the sample tracking monitoring sites can be accurately detected and have no impact on the HLA high-resolution gene detection results. The reference results and the detection results of the tracking monitoring sites are consistent, as shown in Table 4.

[0113] Table 3

[0114]

[0115] Table 4

[0116]

[0117] 2. Verification of the specificity of the reaction solution at the rs3067397 locus:

[0118] The rs3067397 allele-1 reaction solution and the rs3067397 allele-2 reaction solution were respectively used with 3 gDNA samples determined to be negative by sequencing (concentration: 50 ng / μL, from Shanghai Stemir Biotechnology Co., Ltd.) and 1 gDNA sample determined to be positive (a positive sample refers to a sample with a positive detection result for the reaction solution, that is, a sample containing the target sequence detected by the reaction solution) (concentration: 50 ng / μL, from Shanghai Stemir Biotechnology Co., Ltd.). Experiments were carried out according to the implementation method of Example 1. Both reaction solutions could correctly detect the positive samples and there was no non-specific amplification in the negative samples. The amplification curves of the rs3067397 allele-1 reaction solution (upper) and the rs3067397 allele-2 reaction solution (lower) corresponding to the target gene (FAM channel) are shown in Figure 2 .

[0119] 3. Verification of the accuracy of the rs3067397 locus:

[0120] The rs3067397 allele-1 reaction solution and the rs3067397 allele-2 reaction solution were respectively used to detect samples with chimerism rates of 1%, 0.5%, 0.1%, and 0.025% (prepared by mixing negative samples and positive samples in the corresponding proportions. Negative sample concentration: 50 ng / μL, sourced from Shanghai Stemir Biotechnology Co., Ltd.; positive sample concentration: 50 ng / μL, sourced from Shanghai Stemir Biotechnology Co., Ltd.) according to the above RT-qPCR detection procedure. The detection results of both reaction solutions were within the acceptable range. This indicates that the rs3067397 allele-1 reaction solution and the rs3067397 allele-2 reaction solution have good linearity and can accurately detect samples with a chimerism rate as low as 0.025%. The specific results of the rs3067397 allele-1 reaction solution are shown in Table 5, and the specific results of the rs3067397 allele-2 reaction solution are shown in Table 6. The amplification curves of the rs3067397 allele-1 reaction solution (upper) and the rs3067397 allele-2 reaction solution (lower) are shown in Figure 2 , where the blue amplification curve is the amplification curve of the target gene (FAM channel), and the green amplification curve is the amplification curve of the reference gene (VIC channel).

[0121] Table 5

[0122] Sample name Fluorescent channel Detected gene CT value Chimerism rate 1% chimerism rate sample FAM rs3067397 allele-1 26.191 1.366％ 1% chimerism rate sample VIC ALB (reference) 20.648 0.5% chimerism rate sample FAM rs3067397 allele-1 27.059 0.679％ 0.5% chimerism rate sample VIC ALB (reference) 20.582 0.1% chimerism rate sample FAM rs3067397 allele-1 29.613 0.134％ 0.1% chimerism rate sample VIC ALB (reference) 20,754 0.025% chimerism rate sample FAM rs3067397 allele-1 31.605 0.031％ 0.025% chimerism rate sample VIC ALB (reference) 20.637 Positive sample FAM rs3067397 allele-1 22.559 100％ Positive sample VIC ALB (reference) 23.246 Negative sample FAM rs3067397 allele-1 N / A 0 Negative sample VIC ALB (reference) 23.871

[0123] Table 6

[0124]

[0125]

[0126] 4. Repeatability verification of the reaction solution at the rs3067397 locus:

[0127] The rs3067397 allele-1 reaction solution and the rs3067397 allele-2 reaction solution were respectively used to detect a chimeric sample (concentration: 50 ng / μL, sourced from Shanghai Stemir Biotechnology Co., Ltd.) 10 times according to the detection steps of the above post-transplant RT-qPCR detection kit, and the CV value of the logarithmic order of magnitude of the detection results was calculated. The CV value of the logarithmic order of magnitude of the 10 detection results of the rs3067397 allele-1 reaction solution was 4.09% (the specific test results are shown in Table 7); the CV value of the logarithmic order of magnitude of the 10 detection results of the rs3067397 allele-2 reaction solution was 2.00% (the specific test results are shown in Table 8), both meeting the requirements. The amplification curves of the rs3067397 allele-1 reaction solution (upper) and the rs3067397 allele-2 reaction solution (lower) are shown in Figure 2, where the blue amplification curve is the amplification curve of the target gene (FAM channel), and the green amplification curve is the amplification curve of the reference gene (VIC channel).

[0128] Table 7

[0129]

[0130]

[0131] Table 8

[0132]

[0133]

[0134] 5. Detection of cfDNA samples

[0135] Referring to the implementation method of Example 2, 4 positive samples at the rs2307700 locus were detected using the rs2307700 allele-1 reaction solution, and all could be normally detected. The PCR amplification curves are shown in Figure 5 , where the blue amplification curve is the amplification curve of the target gene (FAM channel), and the green amplification curve is the amplification curve of the reference gene (VIC channel).

Claims

1. An allogeneic transplantation tracking and monitoring kit, characterized in that: It comprises a second-generation sequencing detection kit and an RT-qPCR detection kit. The second-generation sequencing detection kit at least comprises a library construction reagent and a recurrence monitoring site capture probe; the recurrence monitoring site capture probe comprises an HLA genotyping detection probe and an HLA high-resolution typing detection supplementary probe.

2. The allogeneic transplantation tracking and monitoring kit according to claim 1, characterized in that: The library construction reagents at least include fragment and end repair solution, fragment and end repair enzyme, ligase, pre-PCR amplification reaction solution, unique double-label primer, purification magnetic beads, capture magnetic beads, post-PCR amplification reaction solution, and post-PCR amplification primer.

3. The allogeneic transplantation tracking and monitoring kit according to claim 1, characterized in that: The RT-qPCR detection kit includes a site reaction solution, a PCR MIX and a negative control.

4. The allogeneic transplantation tracking and monitoring kit according to claim 3, characterized in that: The locus gene frequency in the locus reaction solution is 0.25-0.

75.

5. The allogeneic transplantation tracking and monitoring kit according to claim 3, characterized in that: The number of bases of site insertion and deletion in the site reaction solution is greater than 3.

6. The allogeneic transplantation tracking and monitoring kit according to claim 3, characterized in that: The sites in the site reaction solution include at least one of rs2307700, rs2308010, rs5787309, rs3067397, rs66595817, rs3834231, rs33971783, rs34855933, rs10558392, rs3042783, rs71644113, rs3038530, rs67426579 or the human sex determination gene SRY site.

7. A method for using the allogeneic transplantation tracking and monitoring kit according to any one of claims 2 to 6, characterized in that: The following steps are involved: S1 extracts DNA from pre-transplant recipients and pre-transplant donors; S2: The DNA extracted in step S1 is subjected to PCR amplification using a next-generation sequencing detection kit, and an HLA genotyping detection probe and an HLA high-resolution typing detection supplementary probe are introduced during the PCR amplification process, and a final DNA library is obtained after completion; S3 compares the final DNA library sequencing and uses the sites that the recipient has but the donor does not as the sites for subsequent recurrence monitoring; S4 extracts DNA of the transplant recipient or cfDNA of the transplant recipient; S5: amplify the DNA or cfDNA extracted in step S4 by PCR using an RT-qPCR detection kit; S6 detection, result interpretation and result analysis.

8. The method for using the allogeneic transplantation tracking and monitoring kit according to claim 7, characterized in that: The PCR amplification program of step S5 is 42° C., 5 min; 94° C., 3 min; then 94° C., 15 s, 60° C., 60 s for 40-50 cycles, and the fluorescence signal is collected at the end of the last cycle, and the detection channels are FAM and VIC.

9. The method for using the allogeneic transplantation tracking and monitoring kit according to claim 7, characterized in that: In the result analysis of step S6: if △CT>12, it is determined that the target gene of the recipient before transplantation does not exist in the sample after the recipient allogeneic transplantation; if △CT≤12, it is determined that the target gene of the recipient before transplantation exists in the sample after the recipient allogeneic transplantation, and then quantitative calculation is performed.

10. An application of the allogeneic transplantation tracking and monitoring kit according to any one of claims 1 to 6, characterized in that: Used in hematopoietic stem cell transplantation or solid organ transplantation.

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