Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine / 药物分析学报

Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2-5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90％-110％, and the inter-day and intra-day precision were acceptable (RSD<10％). The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model. Compared to the quantitation methods using UV absorbance, the reported method provides an enhanced sensitivity, and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.

Application of a new magnetic beads made in China in DNA extraction of forensic biological samples with automation workstation / 中国法医学杂志

Objective To study the application of a new magnetic beads made in China in DNA extraction of forensic biological samples with automation workstation.Methods DNA was extracted from common forensic biological samples by QIAGEN Bio-Robert Universal System and a new magnetic beads made in China,and then typed with Identifiler system in ABI3130XL Genetic Analyzer.210 of these samples were also quantitated by ABI7500 Real Time System.Results Total of 9100 genomic DNA was extracted from various forensic biological samples by the new magnetic beads made in China and automation workstation methods,and most of them were successfully typed for STR analysis.In these biological samples,oral swabs and muscles were of the highest Success rate of STR typing(100%),and the lowest was touched cell samples (50.0%).Conclusion The new magnetic beads made in China with automation workstation methods can be applied to DNA extraction of most forensic biological samples.

Clinical Efficacy of Entecavir for Chronic Serious Hepatitis B / 中国药房

OBJECTIVE: To discuss the significance of Entecavir(ETV) in the treatment of chronic serious hepatitis B(HBV).METHODS: 54 patients with chronic serious hepatitis B were assigned to receive combined therapy in which antiviral drugs such as interferon and(or) nucleoside(acid) analogues antiviral drugs were excluded(control group,n=26) or combined therapy in combination with entecavir(0.5 mg?d-1) qd(treatment group,n=28).The course of treatment in both groups were 6 weeks.The hepatic function,HBV markers and HBV-DNA quantitation were deteted every two weeks.The improvement rate of patients after the completion of treatment were recorded.RESULTS: In the follow-up of six weeks,serum HBV-DNA and total bilirubin levels decreased markedly,and significant difference was noted between compared with the control group;ALT,AST,ALB and PT decreased in both groups,but the differnces between the two groups were not significant;there was no signficnant differnce in improvement rate between the treatment group and the control group(89.3% versus 84.6%).CONCLUSION: Entecavir can rapidly lower serum HBV-DNA level,downregulate bilirubin level,improve liver function,improve patients prognosis in patients with hepatitis B,thus it can be used to treat serious hepatitis B.However,used in short term,the survival rate of patients with severe hepatitis B can hardly be improved.

HBV DNA Quantitation Using Real-time PCR / 대한진단검사의학회지

BACKGROUND: Accurate measurement of the concentration of hepatitis B virus (HBV) DNA in clinical samples is important for the appropriate treatment of patients and evaluation of their therapeutic responses. In addition, the concentration of HBV DNA in the serum of patients with chronic HBV infection has a very broad range. Real-time PCR is very sensitive and useful to detect HBV DNA in a wide range of concentrations. We designed new primers and probes for real-time PCR to detect HBV DNA. METHODS: Primers and probes specific for HBV were designed. EUROHEP HBV DNA standards (NIBSC, Hertfordshire, UK) with the HBV DNA concentration of 7.0 x 10(4) copies/mL was used to determine the calibration curve and efficacy for the real-time PCR assay. Sensitivity, dynamic range, and precision were evaluated. The correlation between the real-time PCR and Cobas Amplicor HBV Monitor(TM) assay in the measurement of serum HBV DNA concentrations in 52 patients with chronic HBV infection was evaluated. RESULTS: The sensitivity of the assay was approximately 6.08 x 10(2) copies/mL for HBV, and the quantitation was accurate and reproducible over a wide dynamic range from 6.1 x 0(2) to 6.5 x 10(9) copies/mL without any dilution of specimens. The assay showed low coefficients of variation of repeatability (3.7-24.9%) and reproducibility (7.8-24.7%). The results were found to correlate well with those obtained by Cobas Amplicor HBV Monitor(TM) kit. CONCLUSIONS: We provide a new in-house method for the measurement of serum HBV DNA using real-time PCR, which enables us to detect HBV DNA rapidly, sensitively, and accurately.

The Clinical Significance of Quantitation of HBV DNA in Serum: Comparison of the Branched-DNA Assay with the Second-Generation Digene Hybrid Capture Assay and Long-term Observation / 대한간학회지

BACKGROUNDS/AIMS: Serum levels of hepatitis B virus (HBV) DNA are direct measures of viral replication in hepatocytes. We compared branched-DNA assay (QuantiplexTM, bDNA) and Hybridization Capture II (HCII), and evaluated the clinical significance of HBV DNA quantitation in the natural course of HBV infection. METHODS: We analyzed results of bDNA in 324 serum samples from 83 untreated male patients with chronic hepatitis B. Mean follow up period was 11.8 years. HCII was also performed in 157 arbitrarily selected samples. RESULTS: HBV DNA levels measured with two assays were very similar (r2=0.893, p<0.0001). HBV DNA detection rate of HCII was 6.4% higher than that of bDNA in HBeAg positive samples. HBV DNA detection rate was higher in cases with higher ALT. Among 73 patients with initial diagnosis of chronic hepatitis, 38 patients (52.1%) experienced progression to cirrhosis, and hepatocellular carcinoma (HCC) was developed in 9 (12.3%). HCC was developed in 5 (50.0%) of 10 patients with initial diagnosis of cirrhosis. While HBeAg positive rates during chronic hepatitis, cirrhosis and HCC were 57.8%, 55.0% and 14.3%, respectively (p=0.006), HBV DNA detection rates were 70.6%, 64.0% and 42.9%, respectively (p=0.08). Especially in HCC, the discrepancy between HBeAg positive rate and HBV DNA detection rate may suggest the appearance of variants which cannot produce HBeAg. CONCLUSION: Both HCII and bDNA were similar HBV DNA quantitation assays for clinical use. HBV DNA level correlated with the severity of liver disease. Screening tests for HCC should be recommended in patients whose HBeAg is negative and have detectable HBV DNA.

Comparison of Hybrid Capture System, Hybrid Capture II and Quantiplex HBV DNA Assay for Quantitation of Hepatitis B Virus DNA / 대한임상병리학회지

BACKGROUND: Quantitative measurement of hepatitis B virus (HBV) DNA in serum allows monitoring of HBV replication and assessing responses to antiviral treatment, which is not possible with serological markers for HBV infection. However, most assays developed for the quantitative measurement of HBV DNA have not been standardized. Therefore, we tried to compare the performance of three commercial quantitative methods for the measurement of HBV DNA. METHODS: One hundred consecutive sera with request for HBV DNA quantitation were tested with Hybrid Capture System (HCS), Hybrid Capture II (HC-II) and Quantiplex HBV DNA Assay (bDNA Assay) to evaluate the detection rate, the concordance rate and the correlation of the quantitative results measured by each method. In addition, nested polymerase chain reaction (PCR) was performed for qualitative detection of HBV DNA. RESULTs: Concordance rate was 87% for all three methods, 91% for HCS and HC-II, 94% for HCS and bDNA Assay, and 89% for HC-II and bDNA Assay. HBV DNA quantities measured by three methods showed significant correlation between HCS and HC-II (R=0.88, P<0.0001), HCS and bDNA Assay (R=0.82, P<0.0001), and HC-II and bDNA Assay (R=0.95, P<0.0001). Thirteen sera of discrepant results and 29 of 39 sera of negative results by all three methods showed PCR positivity. CONCLUSIONS: Three quantitative methods for the measurement of HBV DNA showed relatively high concordance rate and good correlation. However, the results by bDNA Assay increased more rapidly than HCS and HC-II as the amounts of HBV DNA in the sample increased that the concentrations of HBV DNA measured by bDNA Assay were two or three times higher than those measured by HCS or HC-II at high HBV DNA concentration range. Thus, further studies are necessary to develop more standardized methods.

DNA Quantitation by Image Cytometry in Bone Tumor / 대한정형외과학회잡지

There are substantial evidence suggesting that DNA content of tumors may provide the prognostic information with independent significances. With the advent of computer and video technology, image analysis becomes a practical method of measuring DNA that also makes it possible to classify cells. Among the patients who were operated and diagnosed as bone tumor at Department of Orthopedic Surgery, Korea University Hospital, from March 1992 to March 1995, we evaluated 19 cases by image cytometry and studied them. Among 19 case, 4 cases were fibrous dysplasia, 2 cases chondroma, 3 cases osteosarcoma, 2 cases chondrosarcoma, and 8 cases were metastatic bone tumors. Total benign tumors were 6 cases and malignant tumors were 13 cases. All benign tumors were diploid and all malignant tumors but one metastatic tumor were aneuploid. As a result, DNA quantitation by image analysis is effective in the differential diagnosis of malignancy in bone tumor. It seems that DNA quantitation will be used on the evaluation of tumor staging and prognosis by further clinical study.

Real time PCR quantificational study of DNA extracted by Chelex-100 method / 中国法医学杂志

Objective To study the relation between the quantity of DNA extracted by Chelex-100 method and multiplex STRs analysis.Methods DNA extracted from a variety of common forensic casework specimens were quantified by using Real-time PCR,and then amplified with AmpFLSTR IdentifilerTM PCR Amplification kit.ResultsAccording to the results of quantification,the quantities of DNA extracted from 113 samples by Chelex-100 method were adjusted to 0.5~3ng for establishing 8?l amplification system,and in this condition,most of 113 forensic casework specimens could be successfully genotyped.Conclusion When the quantity of DNA extracted by Chelex-100 method ranged from 0.5ng to 3ng,most results of multiplex STRs analysis were satisfying.Moreover,the amplification effect of 1?l DNA template was better than 3?l DNA template when the concentrations of extracted DNA were more than 0.5ng/?l.