动物造模 z-bio 2022-05-11 270

　　Objective: The routine immunological detection method of human ABO blood group is not suitable for the detection of experimental monkey blood type, and experimental monkey blood type identification is indispensable in researches such as organ transplantation. Nucleic acid identification method of cynomolgus monkey ABO blood group.

　　Methods: The laboratory established a new molecular detection method for ABO blood type, using competitive allele PCR (KASP) technology, by identifying the bases on the two SNP sites of functional genes closely related to blood type, and pairing them. Rhesus monkeys and cynomolgus monkeys were typed by blood type, and the blood group samples identified by KASP method were identified by Sanger sequencing by designing common PCR primers around the two SNP sites.

　　RESULTS: Fifteen cynomolgus monkey whole blood nucleic acid samples whose blood group had been determined by Taqman PCR method were compared, and the newly established KASP typing results were completely consistent with the original blood type results. The established general PCR method was verified by Sanger sequencing to verify that the KASP method was completely consistent with the sequencing results. In order to verify the repeatability of the newly established KASP method, 6 experimental monkey whole blood with known blood types were selected, and each blood sample was replicated 5 times. The results show that the method has excellent reproducibility. Further, 51 clinical samples were tested by the new KASP method, including 38 samples from cynomolgus monkeys and 13 samples from rhesus monkeys. The blood group results of 2 sites in the same sample were completely Consistent, including 9 blood type A (17.6%), 19 blood type B (37.3%), 23 blood type AB (45.1%), and no blood type O was detected. Among the clinical samples of KASP, 5 samples of each type were selected for general PCR amplification and sequencing analysis, and it was proved that the sequences on the 2 SNP loci were consistent with the KASP typing results.

　　Conclusion: The new KASP blood group identification method has high accuracy and can be used for the rapid detection of ABO blood group in experimental rhesus monkeys and cynomolgus monkeys. Compared with traditional serology or sequencing methods, this method is simple, fast, economical and reliable, and the results are easy At the same time, the requirements for samples are lower than traditional serological methods, and fresh or long-stored whole blood, saliva or nucleic acid can be used for detection.