ABO Genotyping Kit (Real-time PCR method)
SUPER007-008RT-064

The kit is used for the detection of ABO alleles in human red blood cell genomic DNA, including A, B, O, O3, A201, A205 alleles, and could assist in the diagnosis of human red blood cell ABO phenotype. 
The ABO locus is located on human chromosome 9 (9q34), and the A, B, and O genes are highly homologous with 7 exons. The ABO gene is approximately 1060 bp long and encodes 353 amino acids. The A and B genes encode glycosyltransferases, while the O gene is an ineffective gene. There are differences between the B gene and the A gene at four positions: 297, 703, 796, and 803 in the cDNA. The difference between the O gene and the A gene is the deletion of the position 261 base G in cDNA. Type A mainly consists of A1 and A2 subtypes, in addition to A3, Am, and Ax. Compared with the A1 gene, the A2 gene exhibits A/T substitution at the position 467 of cDNA and a deletion of base C at the position 1061. The ABO blood type system is the most important blood type system to ensure the safety of blood transfusions, and ABO blood type identification is also the most critical technology to ensure the safety of blood transfusions. Correct ABO blood type identification is a prerequisite for safe blood transfusion. Importing blood with mismatched ABO blood types can lead to ineffective blood transfusion and even endanger the patient's life. Serological methods for identifying ABO blood group systems can only identify phenotypes and are limited in identifying individuals with abnormal expression and conducting genetic analysis. The ABO blood group genotyping technology can be combined with serological diagnosis results in blood transfusion and applied in clinical. The ABO genotyping results can be used as genetic markers for chimeric detection and subtype research in stem cell transplantation families.

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

---SYBR Green І 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 ABO related gene mutations, deletions and other information, were designed according to the sequence information published in NCBI 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.