RhD Genotyping Kit (Real-time PCR method)
SUPER008-001N-040

The kit is used to predict human RhD blood type and provide reference for RhD blood type identification by detecting the presence of exon 1/3/4/5/6/7/9/10 of the human RhD gene and the 845A and 1227A mutations qualitative. The allele names of the results follow the RhD gene naming principles（v6.2 30-SEP-2022） of the ISBT Red Blood Cell Immunogenetics and Blood Group Naming Committee. The RhD gene encoding RhD blood group antigen is located in the short arm of human chromosome 1, with 10 exons and 9 introns. The length is 57932 bp and it encodes 417 amino acids. Exon deletion, replacement by RHCE gene, and mutations could lead to weaken or deletion of RhD antigens. RhD antigen has strong immunogenicity and is one of the main reasons of hemolytic transfusion response and HDFN.

The kit is based on the PCR-sequence specific primer method (PCR-SSP), and uses multiple sets of allele specific primers to amplify specific RhD 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 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 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 RhD related gene mutations, deletions, insertions and other information, were designed according to the sequence information published in Genbank database of NCBI. 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.