Establishment Of STR Multiplex System And Its Application In Detection Of Chimerism Following Hematopoietic Stem Cell Transplantation

Objective:The purpose of this project is to establish the fluorescence multiplex STR system of D3S1358, D13S317, D7S820, CSF1 PO, D16S539, D12S391, D18S51, D5S818, Amelogenin, FGA. The feasibility of fluorescence multiplex system was explored, which was used in the chimerism detection.Method: According to the population genetics research, D3S1358, D13S317, D7S820, CSF1 PO, D16S539, D12S391, D18S51, D5S818, Amelogenin and FGA were selected. Forward primers of D3S1358, D13S317, D7S820 and CSFIPO were fluorescently labeled by 6-FAM in 5’end, D16S539, D12S391 and D18S51 were labeled by TAMRA in 5’end, Amelogenin, D5S818, FGA were labeled by HEX in 5’end. Reaction conditions such as primer concentration, cycle times, concentration of DNA template, anneal temperature were optimized, to make amplification productions basic balance and specific requirements. To assure the reliability of genotypes, the standard allelic ladder was made by the traditional method.Analysis the results veracity; DNA sensitivity analysis; Analog the mixed chimerism, linear correlation was applied to analyze the results; Repeatability studies; Compared with the sinofiler kit, linear correlation was applied to analyze the results.Result: A fluorescence multiplex system of D3S1358, D13S317, D7S820, CSFIPO, D16S539, D12S391, D18S51, Amelogenin, D5S818, FGA was successfully established. The 25μL Multiplex system: 10′Go Taq Buffer5μL, 2.5mmol/L d NTP3μL, 10μmol/L Primers of D3S1358, D13S317, D7S820, CSF1 PO, D16S539, D12S391, D18S51, Amelogenin, D5S818 and FGA 0.5μL, 0.4μL, 0.7μL, 0.55μL, 0.45μL, 0.5μL, 0.5μL, 0.6μL, 0.7μL, 0.7μL, 10μmol/L Go Taq polymerase 0.3μL, 10ng/μL template DNA 3μL,Water 8.1μL.PCR amplification parameters: 95℃5min;94℃45s,59℃45s,72℃45s,30cycles; 60℃55min;15℃∞. 423 samples genotyping test results wereconsistent with Sinofiler kit. The system could detect the lowest concentration of DNA was 0.4 ng/μL. Analog chimera sample, the minimum detectable donor chimerism rate range was 2.5%-5%. r=0.998, Regression equation: y = 0.9835x+0.7829 R2 = 0.997. Reproducibility experiments, the results showed that the standard deviation was less than 2%, and the intra coefficient of variation was 3.75%, the inter coefficient of variation was 1.23%-7.97%. Contrasted with the results of Sinofiler detection kit, r = 0.997, Regression equation: y = 1.0108x- 0.8418 R2 = 0.9941.Conclusions: A fluorescence labeling multiplex system of D3S1358, D13S317, D7S820, CSF1 PO, D16S539, D12S391, D18S51, Amelogenin, D5S818, FGA was successfully established.This system could be tested by ABI3100, and the genotype was reliable. Donor chimera could be detected by this multiplex system with high sensitivity and strong specificity. Also, this multiplex system was the basis for commercial kit.