Human Leukocytes Antigen Genotyping Kit (Real-time PCR method)
SUPER005-003RT-006
The kit is designed for low resolution genotyping tests of class Ⅰ&Ⅱ of Human Leukocyte Antigen (HLA) alleles of human genome extracted from human’s whole blood for real-time PCR instruments using only. The test of class Ⅰ consists of 3 loci: HLA-A, HLA-B, HLA-C. The test of class Ⅱ consists of 2 loci: HLA-DRB1, HLA-DQB1.

Component 1 of the kit provides genotyping test of HLA-A, HLA-B, HLA-DRB1, HLA-DQB1.

Component 2 of the kit provides genotyping test of HLA-C.
Component 3 of the kit provides genotyping test of HLA-A, HLA-B, HLA-C.

Component 4 of the kit provides genotyping test of HLA-DRB1, HLA-DQB1.

Each component can be used in combination or separately.

Human Leukocytes Antigen (HLA) gene exists on human chromosome 6 and is a double dominant gene. The expressed HLA antigen widely exists on the surface of all cells, however, this antigen was first found on the surface of leukocytes and leukocytes are the most suitable source of material for research, hence called human leukocyte antigen (HLA). Human leukocyte antigen (HLA) is an important immune system for human to identify dissidents. Theoretically, there are almost no two identical individuals. HLA antigens are closely related to the rejection of allogeneic organ transplantation. In allogeneic tissue transplantation, if HLA and other major histocompatibility antigens do not match, the risk of transplant rejection and GVHD will increase^[1-6]. The results of the kit provide clinical reference for physicians in selecting donors and recipients for liver and kidney organ transplantation.

The kit is based on the PCR-sequence specific primer method (PCR-SSP), and uses multiple sets of allele specific primers to amplify specific HLA alleles.

---SYBR Green Ⅰ fluorescent dye used in the amplification reaction solution only emits light after binding with double stranded DNA (dsDNA).

---PCR instrument generates amplification curves and characteristic melting curves to indicate that the amplification is effective by collecting fluorescence signals within the range of maximum absorption wavelength of about 497nm and maximum emission wavelength of about 520nm.

---The positions of positive holes and negative holes are qualitatively judged by the Tm value of the characteristic melting curve.

---Genotyping results,consistent with human genetic rules, shall be qualitatively interpreted according to the combination of positive holes shown in the result typing table.

---In the case of multiple products, only the characteristic melting curve peak of the highest Tm value is displayed.

---Tm value of the characteristic melting curve of the amplification product of the internal reference primer pair was lower than that of the specific amplification product.

---When the reaction of specific primer was negative, Tm value of the internal reference primer pair was detected, revealing that the reaction was effective; When the reaction of specific primer is positive, only the specific amplification product Tm value is displayed.

Specific primers, pointing at HLA related gene mutations, deletions, insertions and other information, were designed according to the sequence information published in EBI, IPD-IMGT/HLA databases. The internal reference primers were designed based on the sequence of the conserved regions of human genes. Internal reference primer pairs are used to check the amplification effectiveness of the reaction systems. They are coated with specific primers in each reaction well to amplify together. At the same time, each plate was individually coated with a well set as a negative control well.