Reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and uses thereof

Abstract

The invention relates to a kit for analyzing chimerism after hemopoietic stem cell is transplanted and the application thereof. The kit contains specific primers containing the following STR loci: D3S3045, D4S2366, D4S2639, D5S818, D13S317, D18S1002, D20S481 and D22S689. The kit adopts primer pairs which are not possessed by the commercialized kit, and the primer pairs provide reagent with higher informedness among recipients, so that the kit is suitable for testing the assembling single information locus and applied for quantitative trace in chimerism.

Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a diagnostic kit for detecting chimerism after hematopoietic stem cell transplantation by using STR and application thereof. Background technique

[0002] Hematopoietic stem cells are one of the components of blood. They are the initial cells that generate various blood cells. They are also called hematopoietic pluripotent stem cells. They exist in bone marrow, embryonic liver, peripheral blood and umbilical cord blood. It not only has a high self-renewal ability, but also has the ability to further differentiate the progenitor cells of various systems. Modern blood transfusions use these two abilities to suppress the immune system of the recipient with radiation or large doses of chemical drugs, and then transfuse the donor's hematopoietic stem cells to allow them to remain in the recipient's bone marrow and differentiate and proliferate. Hematopoietic stem cell transplantation. H...

Examples

Embodiment 1

[0069] Example 1 Screening of STR sites

[0070] Informative analysis of STR loci on Asian hematopoietic stem cell DNA,

[0071] experiment method:

[0072] 1. After obtaining the informed consent of the patients, 2ML of peripheral blood from 16 patients with bone marrow hematopoietic stem cell transplantation and their donors were collected, of which 5 cases were non-blood relationship donor-recipient pairs, and 11 cases were donor-recipient pairs between compatriots right. Genomic DNA was extracted according to the instructions of Tiangen Whole Blood DNA Extraction Kit (Tiangen Company, Beijing, China). After measuring the DNA concentration (nucleic acid and protein detector, Eppendorf, Germany), it was diluted to 10ug / mL, and stored in aliquots.

[0073] 2. Use TAMRA, FAM, and HEX to mark and synthesize different primers (consigned to Shanghai Sangong) for amplification. The amplification sites are shown in Table 1.

[0074] 3. PCR amplification: the amplification syst...

Embodiment 2

[0098] The assembly of embodiment 2 kit

[0099] The primers of SEQ ID NO.1-SEQ ID NO.16 were synthesized respectively.

[0100] For all odd-numbered forward primers, 5’ fluorescently labeled TAMRA or FAM or HEX was used, see Table 1 for details.

[0101] Put different labeled primers into different 0.5mL plastic tubes, seal and protect from light, and put them into the kit to complete the assembly of the kit.

Embodiment 3

[0102] Example 3 Evaluation of the accuracy of the chimerism rate detected by the kit

[0103] experiment method:

[0104] 1. On the basis of the experiment in Example 1, randomly select a sample for the recipient, and determine that the ideal information locus is the D22S689STR locus, the recipient has 16, 17 alleles, and the donor has 13, 20 alleles.

[0105] 2. Both the recipient's DNA and the donor's DNA were placed at 37°C for 30 minutes, and the DNA concentration was pre-adjusted to 10ug / mL.

[0106] 3. Gradient sample preparation Mix the above-mentioned DNA in equal volumes, prepare 50% donor:recipient DNA, and use it as the starting product of the gradient sample, mix it with the donor DNA in turn to form 75%, 87.5%, 93.75%, Gradient samples with chimerism rates of 96.8%, 98.4%, and 99.2%; 50% donor:recipient DNA mixed samples were used as initial samples, which were mixed with recipient DNA before transplantation to form 25%, 12.5%, and 6.25% , 3.13%, 1.56%, 0.8% ch...

technical field

[0001] The invention relates to the field of biotechnology, in particular to a diagnostic kit for detecting chimerism after hematopoietic stem cell transplantation by using STR and application thereof. Background technique

[0002] Hematopoietic stem cells are one of the components of blood. They are the initial cells that generate various blood cells. They are also called hematopoietic pluripotent stem cells. They exist in bone marrow, embryonic liver, peripheral blood and umbilical cord blood. It not only has a high self-renewal ability, but also has the ability to further differentiate the progenitor cells of various systems. Modern blood transfusions use these two abilities to suppress the immune system of the recipient with radiation or large doses of chemical drugs, and then transfuse the donor's hematopoietic stem cells to allow them to remain in the recipient's bone marrow and differentiate and proliferate. Hematopoietic stem cell transplantation. H...

Examples

Embodiment 1

[0069] Example 1 Screening of STR sites

[0070] Informative analysis of STR loci on Asian hematopoietic stem cell DNA,

[0071] experiment method:

[0072] 1. After obtaining the informed consent of the patients, 2ML of peripheral blood from 16 patients with bone marrow hematopoietic stem cell transplantation and their donors were collected, of which 5 cases were non-blood relationship donor-recipient pairs, and 11 cases were donor-recipient pairs between compatriots right. Genomic DNA was extracted according to the instructions of Tiangen Whole Blood DNA Extraction Kit (Tiangen Company, Beijing, China). After measuring the DNA concentration (nucleic acid and protein detector, Eppendorf, Germany), it was diluted to 10ug / mL, and stored in aliquots.

[0073] 2. Use TAMRA, FAM, and HEX to mark and synthesize different primers (consigned to Shanghai Sangong) for amplification. The amplification sites are shown in Table 1.

[0074] 3. PCR amplification: the amplification syst...

Embodiment 2

[0098] The assembly of embodiment 2 kit

[0099] The primers of SEQ ID NO.1-SEQ ID NO.16 were synthesized respectively.

[0100] For all odd-numbered forward primers, 5’ fluorescently labeled TAMRA or FAM or HEX was used, see Table 1 for details.

[0101] Put different labeled primers into different 0.5mL plastic tubes, seal and protect from light, and put them into the kit to complete the assembly of the kit.

Embodiment 3

[0102] Example 3 Evaluation of the accuracy of the chimerism rate detected by the kit

[0103] experiment method:

[0104] 1. On the basis of the experiment in Example 1, randomly select a sample for the recipient, and determine that the ideal information locus is the D22S689STR locus, the recipient has 16, 17 alleles, and the donor has 13, 20 alleles.

[0105] 2. Both the recipient's DNA and the donor's DNA were placed at 37°C for 30 minutes, and the DNA concentration was pre-adjusted to 10ug / mL.

[0106] 3. Gradient sample preparation Mix the above-mentioned DNA in equal volumes, prepare 50% donor:recipient DNA, and use it as the starting product of the gradient sample, mix it with the donor DNA in turn to form 75%, 87.5%, 93.75%, Gradient samples with chimerism rates of 96.8%, 98.4%, and 99.2%; 50% donor:recipient DNA mixed samples were used as initial samples, which were mixed with recipient DNA before transplantation to form 25%, 12.5%, and 6.25% , 3.13%, 1.56%, 0.8% ch...

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