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.

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.

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.

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.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

SMRT sequencing and ddPCR reveal the complexity of developmental trajectories and temporal dynamics of gut bifidobacterial communities in infants

Infant intestinal microbiome is closely linked with health and risk of disease. Bifidobacterium are important components of the infant gut and are known to confer various health effects on the host. However, few studies have described the precise composition and dynamics of early infant gut bifidobacterial communities. Thus, this was a pilot study aiming to describe the developmental trajectories and temporal dynamics of bifidobacterial communities in infants before 6 months of age. A total of 28 fecal samples from 4 infants(GF, ZZ, QM, TN, respectively)were collected and analyzed after 5, 15, 30, 60, 90, 120, 150, and 180 days of birth by a bifidobacteria-target method(based on single-molecule real-time sequencing of partial bifidobacterial rpsK genes)in conjunction with droplet digital polymerase chain reaction(ddPCR). The infant fecal microbiota comprised a total of 11 bifidobacterial species, including 4 major species, i.e., B. dentium(37.35%), B. catenulatum(32.04%), B. breve(22.24%), and B. animalis(8.02%). The infant microbiota showed highly individualized developmental trajectories. The leading species for GF was B. catenulatum, with a relatively stable developmental trajectory. In ZZ, B. breve was enriched, and the developmental trajectory was rather fluctuating. The most abundant species for QM and TN was B. dentium. The developmental trajectory of B. dentium in QM showed a trend of gradual decrease, whereas an opposite trend was seen in samples of TN. The results of ddPCR confirmed large variations in quantities of bifidobacteria between infants and suggested discordances in temporal dynamics of bifidobacterial communities during the first half year of infancy. In conclusion, our results suggested that the early infant gut bifidobacterial microbiota was highly complex and temporal dynamics, with individualized developmental trajectories, which should be considered in future research of infant gut microbiota.

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。

高温胁迫下水稻胚乳淀粉分支酶各同工型基因的表达特征

以稻米品质温度敏感型的早籼稻品种浙辐49为材料,利用人工气候箱控温处理试验和实时荧光定量PCR技术,探讨了水稻发育胚乳中淀粉分支酶(starch branching enzyme,SBE)的3种主要同工型基因(SBEⅠ、SBEⅢ和SBEⅣ)在不同温度处理下的相对表达量差异及动态变化特征。结果表明,高温处理对水稻胚乳中SBE基因的表达调控因其同工型的种类而异,SBEⅣ基因在高温处理下呈上调表达,而SBEⅠ和SBEⅢ基因在高温处理下的相对表达量则明显降低,表现为下调表达;与SBEⅣ等同工型相比,水稻胚乳中SBEⅠ基因对高温胁迫的表达响应相对更敏感。

原发性肝癌患者乙型肝炎病毒标志物模式与病毒DNA载量分析

目的探讨原发性肝癌(PHC)的发生与乙肝标志物(HBV-M)及乙肝病毒DNA(HBV DNA)载量的关系。方法采用化学发光微粒子免疫分析和荧光定量PCR方法,对188例原发性肝癌患者进行HBV-M和HBV DNA检测。结果 PHC患者中,HBsAg(+)、HBeAb(+)、HBcAb(+)组(小三阳)121例,所占比例最多(64.36%),小三阳患者血清HBV DNA检出率为51.23%(62/121),HBV DNA平均拷贝数为104.87±1.67copy/mL;其次为HBsAg(+)、HBeAg(+)、HBcAb(+)组(大三阳)30例(16.1%),患者血清HBV DNA检出率为100%(30/30),HBV DNA平均拷贝数为107.27±1.67copy/mL;再次为HBsAg(+)、HBcAb(+)模式组15例(7.98%),患者血清HBV DNA检出率为46.67%(7/15),HBV DNA平均拷贝数为105.05±1.42copy/mL。HBV DNA检出率小三阳组低于大三阳组,比较差异有统计学意义(P<0.05)。结论 PHC患者HBV感染阳性率高,HBV与PHC有十分密切的关系。

两种方法检测HCV-RNA的结果分析

目的比较两种方法检测丙型肝炎病毒核酸(HCV-RNA)的结果。方法逆转录套式定性PCR(RT-nestedPCR)和荧光定量PCR对288例抗-HCV阳性的丙型肝炎患者血清标本进行HCV-RNA检测。结果288例抗-HCV阳性血清标本有248例RT-nested-PCR结果阳性,阳性率为86.1%(248/288);226例荧光定量PCR检测HCV-RNA>103拷贝/ml,检出率为78.5%(226/288),两种检测结果的阳性符合率为85.9%(219/255)。同时,29例标本定性结果阳性,定量结果<103拷贝/ml;7例标本定性结果阴性,定量结果>103拷贝/ml,差异具有统计学意义(χ2=12.25,P<0.01)。结论两种方法检测HCV-RNA,可以提高检测的灵敏度,为临床治疗提供依据。

多系统萎缩患者TCR α链CDR3谱系的荧光定量PCR溶解曲线

目的:利用荧光定量PCR溶解曲线分析技术检测1例多系统萎缩患者TCRα链CDR3谱系。方法:提取1例多系统萎缩患者和1例正常人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的总RNA,逆转录成cDNA,以32个人TCRα链胚系可变区基因家族(TRAV)设计上游引物,共同的TCRα链恒定区基因家族(TRAC)设计下游引物,荧光定量PCR(FQ-PCR)扩增32个TRAV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单、寡、多克隆增生,挑选单克隆进行PCR扩增,扩增产物行普通琼脂糖凝胶回收后测序分析。结果:多系统萎缩患者和正常人PBMC的32个TCRα链TRAV家族CDR3谱型的FQ-PCR产物的各家族相对定量表达不一;正常人的32个TCRα链TRAV家族CDR3谱型的溶解曲线图表现为多个峰型的高斯分布,多系统萎缩患者的TRAV10、TRAV15、TRAV29家族CDR3谱型的溶解曲线表现为单峰的克隆性增生,其他家族为多峰、寡峰的多克隆及寡克隆增生,未出现缺失的家族,对单峰的TRAV15家族的测序得到的CDR3区的氨基酸组成为:SAIYFCAEDRDSTLTFG。结论:该多系统萎缩患者的PMBC中,存在TCRα链CDR3谱系漂移。

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.

Most common SLC25A13 mutation in 400 Chinese infants with intrahepatic cholestasis

AIM:To establish the real time fluorescence polymerase chain reaction(RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4.METHODS:Four hundred infants(< 1 year of age) with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China were enrolled in this study for detecting their SLC25A13 gene mutation 851del4.Suitable primers and fluorescence-labeled probes for detecting SLC25A13 gene mutation 841del4 were designed.Normal and mutant sequences were detected by PCR with two fluorescence-labeled probes.After a single RT-PCR,results were obtained by analyzing the take-off curves.Twenty-four positive and 14 negative samples were retested by direct sequencing.RESULTS:Eight homozygous and 30 heterozygous mutations were detected in 46 mutant alleles with a 851del4 mutation rate of 5.8%(46/800).Twenty-six and 20 mutant alleles were observed respectively,in 474 and 242 alleles from the intermediate and southern areas of China.No mutant allele was detected in 84 alleles from northern China.Twenty-four positive samples including 4 homozygous and 20 heterozygous mutations,and 14 negative samples were retested by direct sequencing,which confirmed that the accuracy of RTPCR was 100%.CONCLUSION:RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.

Liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients

BACKGROUND Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver disease, but it is not widely used in resource-limited settings. Therefore, non-invasive liquid biopsy tests are needed. AIM To assess liver injury in hepatitis B patients using quantified cell free DNA combined with other serum biomarker as a liquid biopsy-based method. METHODS A cohort of 663 subjects including 313 hepatitis B patients and 350 healthy controls were enrolled. Ultrasound-guided liver biopsies followed by histopathological assessments were performed for the 263 chronic hepatitis B patients to determine the degree of liver injury. Cell-free DNA was quantified using a novel duplex real-time polymerase chain reaction assay. RESULTS Compared with healthy controls, patients with hepatitis B virus (HBV) infection had significantly higher plasma DNA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and HBV DNA levels (P<0.01). Serum ALT, AST, bilirubin, and plasma DNA levels of patients with markedsevere inflammation were significantly higher than those with mild-moderate inflammation (P<0.01). There was a statistically significant correlation between hepatocyte inflammation severity and serum bilirubin (R^2=0.673, P<0.01) or plasma DNA (R^2=0.597, P<0.01) levels. The areas under the curves of serum ALT, bilirubin, plasma DNA, and their combination to distinguish between patients with mild–moderate and marked-severe inflammation were 0.8059, 0.7910, 0.7921, and 0.9564, respectively. CONCLUSION The combination of plasma DNA, serum ALT, and bilirubin could be a candidate liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.

Real-time fluorescent quantitative PCR assay for measuring cytomegalovirus DNA load in patients after haematopoietic stem cell transplantation

Cytomegalovirus （CMV） infection is a major and often deadly complication of haematopoietic stem cell （HSC） transplantation. Successful preemptive CMV therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections. The pp65 antigenemia assay has been used for this purpose with considerable success but has disadvantages of being time-consuming and labor-intensive .