The DNA content and morphometric features of hepatocellular carcinoma (HCC) and liver cell dysplasia (LCD), including nuclear area, nuclear perimeter, nuclear maximum diameter and nuclear circle diameter, were quantit...The DNA content and morphometric features of hepatocellular carcinoma (HCC) and liver cell dysplasia (LCD), including nuclear area, nuclear perimeter, nuclear maximum diameter and nuclear circle diameter, were quantitatively determined by means of image analysis technology. The results showed that in comparison with normal hepatocytes, LCD had a markedly increased DNA content and nuclear morphometric parameters, but the values were lower than those for HCC. LCD showed a slight increase in nuclear atypia represented by the nuclear irregular index, which was also less than HCC. The findings indicate that LCD may be a precaneerous lesion of HCC, to the cells in an abnormal proliferative state.展开更多
To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of ...To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.展开更多
For efficient and quantitative DNA detection,fluorescence staining is the most often explored approach,which relies on non-covalent binding of dyes with double stranded DNA(dsDNA).Ethidium bromide(EB)is the most class...For efficient and quantitative DNA detection,fluorescence staining is the most often explored approach,which relies on non-covalent binding of dyes with double stranded DNA(dsDNA).Ethidium bromide(EB)is the most classic DNA stain,but suffers from its high carcinogenicity.A series of less toxic alternatives were developed,many of which contain the core structure of the benzothiazole ring.However,the relationship between the structure and the DNA detection performance was not illustrated.Herein,five benzothiazole dyes,namely thiazole orange,SYBR Green I,Pico Green,SYBR Safe,and thioflavine-T,were compared for DNA detection through direct fluorescence and gel electrophoresis,with particular focus on the structure-performance relationship.It turned out that SYBR Green I is currently the best choice for DNA detection.The results in this work may be useful for future DNA-staining dye developments.展开更多
Objective: To detect and quantitate genital herpes simplexvirus (HSV) DNA in specimens from 100 patients clinicallydiagnosed with genital herpes. Methods: Polymerase Chain Reaction (PCR) andenzyme-linked immunosorbent...Objective: To detect and quantitate genital herpes simplexvirus (HSV) DNA in specimens from 100 patients clinicallydiagnosed with genital herpes. Methods: Polymerase Chain Reaction (PCR) andenzyme-linked immunosorbent assay (ELISA) were used witha standard curve of DNA copies of HSV as quantitativecontrast. Results: Ninety-three cases were confirmed HSV positiveand 7 cases were found to be negative. There were 58 cases ofHSV-2 (62.4%) and 35 cases of HSV-1(37.6%) among the 93positive cases. The number of DNA plasmids ranged from 115to 1.1×10~5 per 250μL among the 93 positive samples (mean=7.1×10~4/250μL). The number of HSV DNA plasmids rangedfrom 136 to 1.1×10~5 copies per 250μL(mean=7.6×10~4) amongthose with HSV-2, and 115 to 9.4×10~4 per 250μL(mean=6.3×10~4) among those with HSV-1. Meanwhile 10μL ofextracted and dissolved DNA randomly taken from 8 each ofHSV-2 and HSV-1 samples were tested. The number of HSV-2DNA plasmids ranged from 35 copies to 2.7×10~4 (Mean=1.8×10~4) and the number of HSV-1 DNA ranged from 29 to2.5×10~4 (Mean=1.6×10~4). In the 7 negative cases, the quantityof HSV plasmids was zero. Conclusion: The sensitivity of ELISA quantitation (93%) isequal to that of Southern blot. The sensitivity of PCR fordiagnosis is 91%, and 88% for PCR typing.展开更多
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 efficie...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.展开更多
Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognost...Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognostic information for the individual patient. Standard analytical approaches using Southern blot and slot blot require a large amount of good展开更多
A simple method was developed to quantify DNA fragments such as PCR(polymerase chain reaction)products by capillary electrophoresis.Restraint fragments with different lengths were employed as internal standards in the...A simple method was developed to quantify DNA fragments such as PCR(polymerase chain reaction)products by capillary electrophoresis.Restraint fragments with different lengths were employed as internal standards in the study,which makes it possible for the evaluation of the quantity of PCR product in a single run.展开更多
文摘The DNA content and morphometric features of hepatocellular carcinoma (HCC) and liver cell dysplasia (LCD), including nuclear area, nuclear perimeter, nuclear maximum diameter and nuclear circle diameter, were quantitatively determined by means of image analysis technology. The results showed that in comparison with normal hepatocytes, LCD had a markedly increased DNA content and nuclear morphometric parameters, but the values were lower than those for HCC. LCD showed a slight increase in nuclear atypia represented by the nuclear irregular index, which was also less than HCC. The findings indicate that LCD may be a precaneerous lesion of HCC, to the cells in an abnormal proliferative state.
文摘To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.
基金the Major Scientific and Technological Application Program of Chengdu(No.2019-YF09-0081-SN)the Major Scientific and Technological Special Program of Sichuan Province(No.2018SZDZX0027)the Fundamental Research Funds for Central Universities(No.2018SCUH0075)。
文摘For efficient and quantitative DNA detection,fluorescence staining is the most often explored approach,which relies on non-covalent binding of dyes with double stranded DNA(dsDNA).Ethidium bromide(EB)is the most classic DNA stain,but suffers from its high carcinogenicity.A series of less toxic alternatives were developed,many of which contain the core structure of the benzothiazole ring.However,the relationship between the structure and the DNA detection performance was not illustrated.Herein,five benzothiazole dyes,namely thiazole orange,SYBR Green I,Pico Green,SYBR Safe,and thioflavine-T,were compared for DNA detection through direct fluorescence and gel electrophoresis,with particular focus on the structure-performance relationship.It turned out that SYBR Green I is currently the best choice for DNA detection.The results in this work may be useful for future DNA-staining dye developments.
文摘Objective: To detect and quantitate genital herpes simplexvirus (HSV) DNA in specimens from 100 patients clinicallydiagnosed with genital herpes. Methods: Polymerase Chain Reaction (PCR) andenzyme-linked immunosorbent assay (ELISA) were used witha standard curve of DNA copies of HSV as quantitativecontrast. Results: Ninety-three cases were confirmed HSV positiveand 7 cases were found to be negative. There were 58 cases ofHSV-2 (62.4%) and 35 cases of HSV-1(37.6%) among the 93positive cases. The number of DNA plasmids ranged from 115to 1.1×10~5 per 250μL among the 93 positive samples (mean=7.1×10~4/250μL). The number of HSV DNA plasmids rangedfrom 136 to 1.1×10~5 copies per 250μL(mean=7.6×10~4) amongthose with HSV-2, and 115 to 9.4×10~4 per 250μL(mean=6.3×10~4) among those with HSV-1. Meanwhile 10μL ofextracted and dissolved DNA randomly taken from 8 each ofHSV-2 and HSV-1 samples were tested. The number of HSV-2DNA plasmids ranged from 35 copies to 2.7×10~4 (Mean=1.8×10~4) and the number of HSV-1 DNA ranged from 29 to2.5×10~4 (Mean=1.6×10~4). In the 7 negative cases, the quantityof HSV plasmids was zero. Conclusion: The sensitivity of ELISA quantitation (93%) isequal to that of Southern blot. The sensitivity of PCR fordiagnosis is 91%, and 88% for PCR typing.
文摘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.
文摘Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognostic information for the individual patient. Standard analytical approaches using Southern blot and slot blot require a large amount of good
基金This project(29635020)was supported by the National Natural Science Foundation of China.
文摘A simple method was developed to quantify DNA fragments such as PCR(polymerase chain reaction)products by capillary electrophoresis.Restraint fragments with different lengths were employed as internal standards in the study,which makes it possible for the evaluation of the quantity of PCR product in a single run.
