Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

Human papillomavirus 16 physical status detection in preinvasive and invasive cervical carcinoma by multiplex real-time polymerase chain reaction

Objective： To explore an ideal approach for detecting the physical status of HPV-16 in clinic use and to investigate the integrated HPV-16 in CINs and cervical cancer. Methods： Multiplex real-time PCR method was established to quantify the copy numbers of E2 and E6 genes （E2/E6） for analysis of the physical status of HPV-16 DNA and this assay was compared to Southern blot analysis. HPV-16-containing paraffin-embedded tissues including 49 CINs and 51 cervical squamous cancers were detected using the method. Results： （1） The cutoff ratio of E2/E6 to distinguish pure episomal from mixed HPV-16, was 0.81 in the multiplex real-time PCR; （2） The agreement rate between multiplex real-time PCR and Southern blot was 81.5% （the Kappa statistic was 0.844, P〈0.001）; （3） HPV-16 DNA existed in an episomal form in 57.1% and mixed form in 42.9% of CIN I lesions; The concomitant form of HPV-16 （〉70%） constituted the majodty in CIN Ⅱ and CIN Ⅲ; HPV-16 DNA mostly integrated into the host chromosome （s） in squamous cervical cancers （68.6%）; （4） The incidence of HPV-16 integration was increased with the degree of cervical lesions; （5） The frequency of pure integrated HPV-16 in stage Ⅱ＋Ⅲ （88%） was significantly higher than that in stage Ⅰ （33.3%）. Conclusion： （1） Mutiplex real-time PCR provides a rapid, sensitive and reliable method for clinic detection of the physical state of HPV-16 DNA; （2） The integration of the HPV-16 DNA is a very eady and important event in the progression from preinvasive to invasive cervical cancer; （3） The pure integrated status of HPV-16 in cervical cancer may be associated with poor prognosis of cervical cancer, but further study will be needed to prove its prognostic significance.

Expression of cellular fibronectin mRNA in adult periodontitis and peri-implantitis: a real-time polymerase chain reaction study

Cellular fibronectin （cFn） is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA （mRNA） in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction （PCR） and to determine its clinical significance. A total of 30 patients were divided into three groups of 10： healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies （1 mmx I mmx I mm） from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group （1.526＋0.441） was lower than that in the healthy group （3.253＋0.736）. However, the mRNA expression of cFn in the peri-implantitis group （3.965＋0.537） was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.

Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis

AIM： To design, optimize and validate a rapid,internally controlled real-time polymerase chain reaction（RT-PCR） test for herpes simplex virus（HSV） in the diagnosis of necrotizing herpes stromal keratitis.· METHODS： Tears alone or together with corneal epithelium scrapings from 30 patients（30 eyes）suspected of necrotizing herpes stromal keratitis were tested for HSV DNA by RT-PCR. The samples were collected during the first visit and then on the subsequent 7, 14, 28, 42, and 56 d. The symptoms of the patients were scored before treatment to determine the correlation between HSV concentration in the corneal epithelium scrapings and clinical scores.·RESULTS： The positive rate（46.4%） in the corneal epithelium group before the therapy was significantly higher than that（13.3%） in the tears group（P =0.006）.There were 13 positive HSV patients before the therapy,the concentration of HSV DNA in corneal epithelium scrapings group was significantly higher than that in the tears group（paired t-test, P =0.0397）. Multilevel mixedeffects model analysis showed that the difference between the corneal epithelium scrapings group and the tears group was statistically significant（P =0.0049）. The Spearman rank correlation analysis indicated a positive correlation between the HSV concentration in the corneal epithelium scrapings and clinical scores before the treatment（r =0.844, P〈 0.0001）.· CONCLUSION： RT-PCR appears to be a powerful molecular tool for the diagnosis of necrotizing herpes stromal keratitis.

Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis

AIM: To develop a real-time reverse transcriptionpolymerase chain reaction(RT-PCR) assay to genotype rotavirus(G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR(rt-g PCR) by selecting genotype-specific primers of published conventional RT nested PCR(cn RT-PCR) assay and optimizing the amplification conditions. c DNA was first synthesized from total RNA with Super Script? Ⅱ reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cn RT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gelelectrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January2012 and June 2013.RESULTS: The new rt-g PCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 ℃ to 82 ℃. The sensitivity of rt-g PCR was comparable to cn RT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-g PCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8](42.6%), G2P[4](4.9%), G3P[8](10.7%), G9P[8](10.7%), G9P[4](6.6%), G12P[8](23.0%), and unknown GP[8](0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool(http://rotac.regatools.be/). All genotyping results were confirmed to be correct.CONCLUSION: rt-g PCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.

Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

Analysis of hepcidin expression: In situ hybridization and quantitative polymerase chain reaction from paraffin sections

AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). METHODS: Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. RESULTS: Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. CONCLUSION: We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Use of real-time polymerase chain reaction for the diagnosis of Pneumocystis pneumonia in immunocompromised patients： a meta-analysis

Background The diagnosis of Pneumocystis pneumonia （PCP） in immunocompromised patients is still challenging today due to the absence of an in vitro culture system and the low diagnostic accuracy of microscopic examinations. Herein, we performed a meta-analysis to evaluate the accuracy of real-time polymerase chain reaction （PCR） in the diagnosis of PCP. Methods We searched Web of Knowledge and Medline from 1990 to May 2010 for studies reporting diagnostic accuracy data regarding the use of real-time PCR in the diagnosis of PCP in immunocompromised patients. Results Ten individual studies were included. Overall, the sensitivity of real-time PCR was 97% （95% CI： 93%-99%）; the specificity was 94% （95% CI： 90%-96%）. The area under the HSROC curve （95% CO for real-time PCR was 0.99 （0.97-0.99）. In a subgroup analysis regarding studies involving HIV patients among the study population, the sensitivity and specificity were 97% （95% CI： 93%-99%） and 93% （95% CI： 89%-96%）, respectively. Regarding studies using Bronchoalveolar lavage （BAL） samples only： sensitivity =98% （95% CI： 94%-99%）; specificity =93% （95% CI： 89%- 96%）, respectively. Regarding studies using microscopy as a reference standard： sensitivity =97% （95% CI： 92%-99%）; specificity =93% （95% CI： 88%-96%）. However, high between-study statistical heterogeneity was observed in all analyses. Conclusions Real-time PCR has a good diagnostic accuracy and may provide a useful adjunctive tool for the diagnosis of PCP in immunocompromised patients. Further studies are needed in order to identify any differences in the diagnostic performance of real-time PCR in HIV and non-HIV immunocompromised patients.

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

Comparison of Abbott and Da-an real-time PCR for quantitating serum HBV DNA

AIM: To compare the performance of the Da-an real-time hepatitis B virus (HBV) DNA assay and Abbott RealTime HBV assay.

实时荧光定量PCR定量方法研究进展

实时荧光定量PCR以其特异性强、灵敏度高、重复性好、定量准确、速度快、全封闭反应等优点而成为了分子生物学研究中的重要工具,综述了实时荧光定量PCR技术及其定量方法的研究进展,并展望了其应用前景。

实时荧光聚合酶链反应技术在献血者血液筛查中的应用

目的评估将实时荧光聚合酶链反应技术应用于血液常规筛查的必要性。方法使用实时荧光聚合酶链反应技术对酶免双试剂及转氨酶检测合格的无偿献血者血液标本进行核酸筛查，初筛阳性标本进行病毒项目确认。确认阳性标本进一步做病毒载量和血清标志物检测。结果19562人份酶免阴性标本中共检出12份HBVDNA阳性标本，病毒载量为（〈12～30．6）IU／mL。其中5份为乙肝隐匿性感染，3份为乙肝感染恢复期，2份为变异病毒感染，2份为疑似乙肝窗口期感染。结论将实时荧光聚合酶链反应技术应用于血液筛查有利于提高成都地区输血安全，特别是对检出ELISAHBsAS阴性的低浓度乙型肝炎感染者具有重要意义。

实时荧光聚合酶链反应检测HBV基因型B～D方法的建立

目的建立实时荧光聚合酶链反应检测乙型肝炎病毒(HBV)基因型B^D方法并验证其准确性。方法比较GenBank中已发表的明确分型的143株HBV全序列,设计特异的组合引物探针,应用实时荧光聚合酶链反应HBV基因分型法对兰州地区128例慢性乙型肝炎患者血清标本进行基因型B、C、D检测,并随机对检测的基因型各3株标本进行S基因测序验证。结果128例标本中B型检出率为20.3%(26/128),C型71.9%(92/128),D型7.8%(10/128);18株HBV克隆标本S基因测序结果与本分型法完全一致。结论实时荧光聚合酶链反应HBV基因分型法能够简便、灵敏、快速、准确地鉴定HBV基因型,适宜用于HBV基因型的大规模临床和流行病学调查。

Update on occult hepatitis B virus infection

The event of mutations in the surface antigen gene of hepatitis B virus(HBV) results in undetectable hepatitis B surface antigen with positive/negative anti-hepatitis B core(anti-HBc) antibody status in serum and this phenomenon is named occult hepatitis B infection(OBI). The presence of anti-HBc antibody in serum is an important key for OBI tracking, although about 20% of OBI cases are negative for anti-HBc antibody. The diagnosis of OBI is mainly based on polymerase chain reaction(PCR) and real-time PCR assays. However, real-time PCR is a more reliable method than PCR. OBI is a great issue for the public health problem and a challenge for the clinical entity worldwide. The persistence of OBI may lead to the development of cirrhosis and hepatocellular carcinoma. With regard to OBI complications, the screening of HBV DNA by the highly sensitive molecular means should be implemented for:(1) patients with a previous history of chronic or acute HBV infection;(2) patients co-infected with hepatitis C virus/human immunodeficiency virus;(3) patients undergoing chemotherapy or anti-CD20 therapy;(4) recipients of organ transplant;(5) blood donors;(6) organ transplant donors;(7) thalassemia and hemophilia patients;(8) health care workers;(9) patients with liver related disease(cryptogenic);(10) hemodialysis patients;(11) patients undergoing lamivudine or interferon therapy; and(12) children in time of HBV vaccination especially in highly endemic areas of HBV. Active HBV vaccination should be implemented for the close relatives of patients who are negative for OBI markers. Thus, the goal of this review is to evaluate the rate of OBI with a focus on status of high risk groups in different regions of the world.

Expression of matrix metalloproteinases 2 and 9 in human gastric cancer and superficial gastritis

AIM:To assess expression of matrix metalloproteinases 2(MMP2)and MMP9 in gastric cancer,superficial gastritis and normal mucosa,and to measure metalloproteinase activity.METHODS:MMP2 and MMP9 mRNA expression was determined by quantitative real-time polymerase chain reaction.Normalization was carried out using three different factors.Proteins were analyzed by quantitative gelatin zymography(qGZ).RESULTS:18S ribosomal RNA(18SRNA)was very highly expressed,while hypoxanthine ribosyltransferase-1(HPRT-1)was moderately expressed.MMP2 was highly expressed,while MMP9 was not detected or lowly expressed in normal tissues,moderately or highly expressed in gastritis and highly expressed in cancer.Relative expression of 18SRNA and HPRT-1 showed no significant differences.Significant differences in MMP2 and MMP9 were found between cancer and normal tissue,but not between gastritis and normal tissue.Absolute quantification of MMP9 echoed this pattern,but differential expression of MMP2 proved conflictive.Analysis by qGZ indicated significant differences between cancer and normal tissue in MMP-2,total MMP-9,250 and 110 kDa bands.CONCLUSION:MMP9 expression is enhanced in gastric cancer compared to normal mucosa;interpretation of differential expression of MMP2 is difficult to establish.

Involvement of interstitial cells of Cajal in experimental severe acute pancreatitis in rats

AIM:To observe the changes in interstitial cells of Cajal(ICC) in rats with experimental severe acute pancreatitis(SAP).METHODS:A total of twenty-four SD rats were randomly divided into two groups(n = 12),namely the sham(S) group and the SAP group;the SAP rat model was established by retrograde injection of 5% sodium taurocholate(1.0 mL/kg) into the pancreatic duct.Twenty-four hours later intestinal motility was assessed by testing small intestinal propulsion rate,and then the rats were sacrificed.The pancreas and jejunum were resected and underwent routine pathologic examination.Immunohistochemical staining was used to detect c-kit-positive cells in the jejunum.Expression of c-kit mRNA was detected by real-time polymerase chain reaction,and the expression of c-kit protein was evaluated by Western blotting.Ultrastructure of ICC was evaluated by transmission electron microscopy.RESULTS:There was bleeding,necrosis and a largeamount of inflammatory cell infiltration in pancreatic tissue in the SAP group,while in jejunal tissue we observed a markedly denuded mucosal layer,loss of villous tissue and a slightly dilated muscular layer.The small intestinal propulsion rate was 68.66% ± 2.66% in the S group and 41.55% ± 3.85% in the SAP group.Compared with the S group,the rate of the SAP group decreased sharply.The density of c-kit-positive cells in the SAP group was significantly lower than in the S group;the respective mean densities were 88.47 ± 10.49 in the S group and 56.11 ± 7.09 in the SAP group.The levels of c-kit protein and mRNA were 0.36 ± 0.04 and 1.29 ± 0.91 in the SAP group,respectively,which were significantly lower than those in the S group(0.53 ± 0.06,0.64 ± 0.33,respectively).In the SAP group,ICC profiles showed the same change tendency,such as vacuolation of mitochondria,irregular vacuoles and loosened desmosome-like junctions.CONCLUSION:Decreased c-kit-positive cells and ultrastructural changes in ICC resulting from blockade of the c-kit signaling pathway are involved in the intestinal dysmotility associated with SAP.

Influence of efflux pump inhibitors on the multidrug resistance of Helicobacter pylori

AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's blood. The multidrug resistant (MDR) H. pylori were obtained with the inducer chloramphenicol by repeated doubling of the concentration until no colony was seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present study included periods before and after the advent of the EPIs, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine and pantoprazole), and the minimum inhibitory concentrations (MICs) were determined accordingly. In the same way, the effects of 5 proton pump inhibitors (PPIs), used in treatment of H. pylori infection, on MICs of antibiotics were evaluated.RESULTS: Four strains of MDR H. pylori were induced successfully, and the antibiotic susceptibilities of MDR strains were partly restored by CCCP and pantoprazole, but there was little effect of reserpine. Rabeprazole was the most effective of the 5 PPIs which could decrease the MICs of antibiotics for MDR H. pylori significantly.CONCLUSION: In vitro, some EPIs can strengthen the activities of different antibiotics which are the putative substrates of the efflux pump system in H. pylori.

Clinical significance of serum miR-21 in breast cancer compared with CA153 and CEA

Objective： MicroRNA-21 （miR-21） has been shown to be a key regulator of carcinogenesis. There were few reports about the comparison of serum miR-21 with conventional tumor markers. This study aimed to explore the diagnostic value of circulating miR-21 as a tumor marker in breast cancer （BC） and compare it with CA15 3 and carcinoembryonic antigen （CEA）. Methods： Circulating miR-16 and miR-21 were amplified and quantitatively detected by real-time PCR in 89 BC patients and 55 healthy controls. The levels of CA153 and CEA were measured through assays. Then the sensitivity in diagnosis of BC was compared among miR-21, CA153 and CEA. Results： The level of serum miR-21 was significantly higher in BC patients than controls （P〈0.001）. The sensitivity and specificity of miR-21 were 87.6% and 87.3%, respectively, whereas the sensitivities of CEA and CA153 were only 22.47% and 15.73%. Con^lusions： Compared with CEA and CA153, serum miR-21 has a higher sensitivity in diagnosis of BC. Although not correlated with the status of ER, PR and clinical stages, serum miR-21 may be a potential diagnostic indicator for BC, especially for the early stage.