Objective To establish the allele-specific real-time polymerase chain reaction (ASPCR) for detection of neonatal hyperbilirubinemia related gene SLCO1B1 A388G polymorphism and apply this assay to identify the clinical samples.Methods According to SLCO1B1 A388G polymorphism loci,specific primers were designed and the assay was established.Wide type plasmid and mutant plasmid were constructed.Fifty clinical samples were selected,including 30 samples of neonatal hyperbilirubinemia that had been diagnosed with SLCO1B1 A388G mutant and 20 samples of healthy newborns without SLCO1B1 A388G mutant were selected as the controls.Wide type plasmid,mutant plasmid and clinical samples were tested by specific and non-specific primers.A388G polymorphism was determined by difference in Ct (cycle threshold) between specific and non-specific primers.Then,the accuracy,sensitivity and specificity of assay were evaluated.Results The difference in Ct (cycle threshold) between specific and non-specific primers that amplified equivalent wide type template was 13.97 ±0.75.The assay could correctly distinguish the wide type and mutant plasmid.Probit regression analysis showed the sensitivity of the assay could reach to 5.28 copies/μL.For clinical samples,the Ct values of the samples with A388G mutation was less than 37.75 and showed positive results,while the samples without A388G mutation did not show any amplification nor Ct values were larger than 37.75,which showed negative results.Conclusions ASPCR is a fast,simple and effective method for SLCO1B1 A388G polymorphism detection of the clinical simples.It can be used for large sample screening for neonatal hyperbilirubinemia gene loci.

Objective To investigate the relationship between the methylation status of mucoprotein 3A (MUC3A)gene and the prognosis of children with neuroblastoma(NB).Methods A pathological section of 92 cases of NB from January 2013 to December 2015 in Department of Pathology,Children and Women′s Healthcare Hospital of Laiwu City,were co-llected.There were 43 males and 49 females;and their ages ranged from 3 months to 13 years old, in which 31 cases were less than 1 year old,and 61 cases were over 1 year old.Methylation specific polymerase chain reaction was used to detect the methylation status of MUC3A gene in 92 cases of NB,and the relationship between the methylation status of MUC3A gene and the clinicopathological features of NB were analyzed.According to the methyla-tion status of MUC3A gene,the 3-year survival rate and survival time of the MUC3A gene methylation positive group and the negative group were compared.Results For those over 1 year old,the pathological types were differentiated +undifferentiated and in clinical stage of stage Ⅲ+Ⅳ,their MYCN gene amplification,MUC3A gene methylation inci-dence(86.89%,91.80%,90.32%,81.82%)were significantly higher than those less than 1 year old,whose patho-logical types were well differentiated and in clinical stage of Ⅰ + Ⅱ,MYCN gene was not amplified(38. 71%, 29.03%,30. 00%,42.31%),and the differences were statistically significant(χ2= 23.007,39. 059,35. 480, 14.043,all P<0.001).The 3 year survival rate of MUC3A gene methylation positive children(26.15%)was signifi-cantly lower than that of MUC3A gene methylation negative children(66.67%),and the difference was statistically sig-nificant(χ2=13.283,P<0.001).The median survival time of MUC3A gene methylation positive children in 3-year old children was 19 months,significantly shorter than that of MUC3A gene methylation negative children(32 months), and the difference was statistically significant(log-rank:χ2=5.910,P=0.015).Conclusion The positive methyla-tion of MUC3A gene suggests that children with NB have poor prognosis.