A technique has been developed using PCR to detect monoclonality of B-lymphoproliferative disorders. DNA was extracted from the blood, tissue and paraffin embedded sections by biochemical means or boiling. Forty cases...A technique has been developed using PCR to detect monoclonality of B-lymphoproliferative disorders. DNA was extracted from the blood, tissue and paraffin embedded sections by biochemical means or boiling. Forty cases of B-non Hodgkin's lymphoma (NHL), 15 cases of T-NHL, 8 cases of chronic lymphocytic leukemia, 17 cases of reactive lymphadenopathy and 12 cases of various non-lym-phocytic tumor were examined. Monoclonality of B-lymphocytes was detected in 86-92% of cases with B-lymphoproliferative diseases, but none in T-NHL, reactive disorders and non-lymphatic tumors. This technique provides a new molecular biologic method to diagnose malignant B-lymphoproliferative dicor-ders. It may be useful in Ig gene rearrangement study, differential diagnosis and retrospective investigation of lymphoproliferative disorders.展开更多
Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected...Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.展开更多
Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Und...Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction（q PCR） based on 18 S r DNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression（R2 = 0.91） was generated based on cycle thresholds value（Ct） versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using q PCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abundance, category 3 brown tide blooms（〉200 000 cells/m L） occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms（35 000–200 000 cells/m L） in August and all stations had category 1 blooms（〉0 to ≤35 000 cells/m L） in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.展开更多
Polymerase chain reaction (PCR) was used to detect the presence of Barrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA seq...Polymerase chain reaction (PCR) was used to detect the presence of Barrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridization with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdorferi genome, even in the presence of a 104-fold excess of human eukaryotic DNA , and was also specific to different B. burgdorferi strains tested. Sera serologically positive to B. burgdcrferi (n=26), bronchoalveolar lavage fluid and supernatant of BALF (n=26) and peripheral blood (n=9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26. and 0/9, respectively). It was considered that DNA from B. burgdor ferimay be identified in a minority of patients with sarcoidosis, and it may play a pathogenetic role in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.展开更多
Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher ampli...Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 3 hr at 30°C or 1 hr at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentration, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not primarily due to the reduced polymerase concentration resulted from the above interaction. This study provided another good start to investigate potential implications of quantum dots in key molecular biology techniques.展开更多
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 e...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.展开更多
A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tiv...A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tivity assay of PCR detection was performed by making dilutions of CPV positive DNA extracted from fecal sample, carrying out PCR for each dilution and visualiz-ing amplicons in ethidium bromide stained agarose gel under UV radiation. Study was valuable in determining the efficiency of PCR. The sensitivity of PCR in present study was determined to be equivalent to detection of .00 2pg/μl of CPV DNA. The study was conducted to analyze the variation, sensitivity and repeatability.展开更多
Using polymerase chain reaction (PCR), 25 specimens from 22 patients with oral carcinomas were examined by 6 selected primers of human papillomavirus (HPV). Eighteen of the 22 patients (18/22) gave positive reaction w...Using polymerase chain reaction (PCR), 25 specimens from 22 patients with oral carcinomas were examined by 6 selected primers of human papillomavirus (HPV). Eighteen of the 22 patients (18/22) gave positive reaction with a positive rate of 81.8%. The positive rates of HPV 6, 11, 16 and 18 were 27.3%, 18.2%, 63.6% and 40.9% respectively. 13.6% positive for mixed infection of HPV 16 and 18 (3/22) and 18.2% positive for mixed infection of HPV, 6, 11, 16 and 18 (4/22). Examining enlarged cervical lymph nodes in three cases with suspecting metastases to cervical lymph nodes from oral carcinomas. It revealed HPV DNA 16 and 18 in two cases and HPV DNA 18 in one case. These results suggested that there was a tendency for HPV 16 and 18 to metastasinze via lymphatics. Only one case of the three had a pathologic diagnosis of lymph node metastasis. Of the 30 non tumor controls, HPV DNA positivity was 10%, all being HPV 18. χ 2 test gave a P<0.005. It strongly indicated that HPV 16 and 18 were related to oral carcinomas.展开更多
文摘A technique has been developed using PCR to detect monoclonality of B-lymphoproliferative disorders. DNA was extracted from the blood, tissue and paraffin embedded sections by biochemical means or boiling. Forty cases of B-non Hodgkin's lymphoma (NHL), 15 cases of T-NHL, 8 cases of chronic lymphocytic leukemia, 17 cases of reactive lymphadenopathy and 12 cases of various non-lym-phocytic tumor were examined. Monoclonality of B-lymphocytes was detected in 86-92% of cases with B-lymphoproliferative diseases, but none in T-NHL, reactive disorders and non-lymphatic tumors. This technique provides a new molecular biologic method to diagnose malignant B-lymphoproliferative dicor-ders. It may be useful in Ig gene rearrangement study, differential diagnosis and retrospective investigation of lymphoproliferative disorders.
基金supported by a research grant from the Office of the Vice-Chancellor for Research and Development,University of the Philippines-Diliman(Grant No.101007 PNSE)to W.L.R.and H.J.S
文摘Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.
基金The National Natural Science Foundation of China under contract No.40906081the Public Science and Technology Research Funds Projects of Ocean under contract Nos 2011418021 and 201305030Integrated Environmental Governances and Restoration in Beidaihe Coast of Hebei Province
文摘Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction（q PCR） based on 18 S r DNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression（R2 = 0.91） was generated based on cycle thresholds value（Ct） versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using q PCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abundance, category 3 brown tide blooms（〉200 000 cells/m L） occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms（35 000–200 000 cells/m L） in August and all stations had category 1 blooms（〉0 to ≤35 000 cells/m L） in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.
文摘Polymerase chain reaction (PCR) was used to detect the presence of Barrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridization with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdorferi genome, even in the presence of a 104-fold excess of human eukaryotic DNA , and was also specific to different B. burgdorferi strains tested. Sera serologically positive to B. burgdcrferi (n=26), bronchoalveolar lavage fluid and supernatant of BALF (n=26) and peripheral blood (n=9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26. and 0/9, respectively). It was considered that DNA from B. burgdor ferimay be identified in a minority of patients with sarcoidosis, and it may play a pathogenetic role in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.
文摘Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 3 hr at 30°C or 1 hr at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentration, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not primarily due to the reduced polymerase concentration resulted from the above interaction. This study provided another good start to investigate potential implications of quantum dots in key molecular biology techniques.
文摘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.
文摘A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tivity assay of PCR detection was performed by making dilutions of CPV positive DNA extracted from fecal sample, carrying out PCR for each dilution and visualiz-ing amplicons in ethidium bromide stained agarose gel under UV radiation. Study was valuable in determining the efficiency of PCR. The sensitivity of PCR in present study was determined to be equivalent to detection of .00 2pg/μl of CPV DNA. The study was conducted to analyze the variation, sensitivity and repeatability.
文摘Using polymerase chain reaction (PCR), 25 specimens from 22 patients with oral carcinomas were examined by 6 selected primers of human papillomavirus (HPV). Eighteen of the 22 patients (18/22) gave positive reaction with a positive rate of 81.8%. The positive rates of HPV 6, 11, 16 and 18 were 27.3%, 18.2%, 63.6% and 40.9% respectively. 13.6% positive for mixed infection of HPV 16 and 18 (3/22) and 18.2% positive for mixed infection of HPV, 6, 11, 16 and 18 (4/22). Examining enlarged cervical lymph nodes in three cases with suspecting metastases to cervical lymph nodes from oral carcinomas. It revealed HPV DNA 16 and 18 in two cases and HPV DNA 18 in one case. These results suggested that there was a tendency for HPV 16 and 18 to metastasinze via lymphatics. Only one case of the three had a pathologic diagnosis of lymph node metastasis. Of the 30 non tumor controls, HPV DNA positivity was 10%, all being HPV 18. χ 2 test gave a P<0.005. It strongly indicated that HPV 16 and 18 were related to oral carcinomas.
