5-hydroxymethylcytosine(5hmC)is a recently discovered epigenetic modification which is widespread in many cell types and tissues.The dynamic variation of abundance may indicate physiological and pathological changes.Since 5hmC is a potential marker in cancer early diagnosis,site specific detections should be developed more than global level quantification.The detection of site specific 5hmC has been hampered by the lack of distinction between 5-methylcytosine(5mC)and 5hmC.Both the two bases resist deamination during traditional bisulfite treatment.Thus,we cannot design suitable probes or primers to detect them individually.In this work,Tet assisted bisulfite(TAB)[1] treatment was used.We protected 5hmC through glucosylation,and then oxidized 5mC to 5caC by Tet.After subsequent bisulfite conversion,the 5caC and cytosine(C)were converted to uracil(U),whereas β-glucosyl-5-hydroxymethylcyrosine(5gmC)from 5hmC was reserved for quantification.Using PCR primers,specific probes and FRET hairpins,multiplex 5hmC markers were detected simultaneously in-one-well by real-time dual-amplification quantification(RTDAQ).The strategy combined PCR-based exponential amplification and FRET hairpin based linear signal amplification.It can detect as low as 60 copies single base 5hmC site.Despite the reduced genomic complexity after bisulfite conversion,the method illustrated high specificity.For high density 5hmC site,0.002%5hmC marker spiking in C/5mC/5hmC mixture was detectable.For single base 5hmC site,the specificity was 99%,which is enough to distinguish 5hmC/5mC and quantify accurately when analyzing the gDNA extracted from tissue or blood samples.The TAB-5hmC-RTDAQ method has potential to be a powerful tool for the detection of site specific 5hmC markers in clinical diagnosis.