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Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction 被引量:25
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作者 Wei-Ping Qian Yue-Qiu Tan +7 位作者 Ying Chen Ying Peng Zhi Li Guang-Xiu Lu Made C. Liu Hsiang-Fu Kung Ming-Ling He Li-Ka Shing 《World Journal of Gastroenterology》 SCIE CAS CSCD 2005年第34期5385-5389,共5页
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 carr... 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. 展开更多
关键词 Hepatitis B virus SEMEN real-time polymerase chain reaction Viral load
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Human papillomavirus 16 physical status detection in preinvasive and invasive cervical carcinoma by multiplex real-time polymerase chain reaction 被引量:5
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作者 Ying Zheng Zhilan Peng Jiangyan Lou He Wang 《The Chinese-German Journal of Clinical Oncology》 CAS 2007年第1期72-79,共8页
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 est... 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. 展开更多
关键词 HPV multiplex real-time polymerase chain reaction INTEGRATION cervical carcinoma
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Expression of cellular fibronectin mRNA in adult periodontitis and peri-implantitis: a real-time polymerase chain reaction study 被引量:1
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作者 Yan-Yun Wu Huan-Huan Cao +2 位作者 Ning Kang Ping Gong Guo-Min Ou 《International Journal of Oral Science》 SCIE CAS CSCD 2013年第4期212-216,共5页
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 ... 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. 展开更多
关键词 adult periodontitis cellular fibronectin PERI-IMPLANTITIS real-time polymerase chain reaction
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Real-time polymerase chain reaction for the diagnosis of necrotizing herpes stromal keratitis 被引量:1
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作者 Jun-Xin Ma Lin-Nong Wang +2 位作者 Ru-Xia Zhou Yang Yu Tong-Xin Du 《International Journal of Ophthalmology(English edition)》 SCIE CAS 2016年第5期682-686,共5页
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.· M... 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. 展开更多
关键词 necrotizingherpes stromal keratitis real-time polymerase chain reaction corneal epithelium scrapings TEARS
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Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis 被引量:1
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作者 Yupin Tong Bonita E Lee Xiaoli L Pang 《World Journal of Virology》 2015年第4期365-371,共7页
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 approa... 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. 展开更多
关键词 ROTAVIRUS A Melting temperature real-time polymerase chain reaction SYBR green GENOTYPING
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Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression 被引量:1
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作者 Bing Li Zhaozhe Liu Xiaodong Xie Yakun Wang 《The Chinese-German Journal of Clinical Oncology》 CAS 2010年第6期316-320,共5页
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
关键词 breast cancer Metadherin (MTDH) real-time fluorescence quantitative polymerase chain reaction (PCR)
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Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats
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作者 Muhammad Zulfiqah Sadikan Nurul Alimah Abdul Nasir +2 位作者 Mohammad Johari Ibahim Igor Iezhitsa Renu Agarwal 《International Journal of Ophthalmology(English edition)》 SCIE CAS 2024年第5期794-805,共12页
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 trans... 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. 展开更多
关键词 housekeeping genes stability real-time reverse transcription polymerase chain reaction retinal tissue streptozotocin-induced diabetic rats
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Analysis of hepcidin expression: In situ hybridization and quantitative polymerase chain reaction from paraffin sections 被引量:1
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作者 Yuhki Sakuraoka Tokihiko Sawada +4 位作者 Takayuki Shiraki Kyunghwa Park Yuhichiro Sakurai Naohisa Tomosugi Keiichi Kubota 《World Journal of Gastroenterology》 SCIE CAS CSCD 2012年第28期3727-3731,共5页
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 R... 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. 展开更多
关键词 HEPCIDIN EXPRESSION In situ hybridization IMMUNOHISTOCHEMISTRY real-time polymerase chain reaction
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Use of real-time polymerase chain reaction for the diagnosis of Pneumocystis pneumonia in immunocompromised patients: a meta-analysis 被引量:6
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作者 Hanssa Summah ZHU Ying-gang +2 位作者 Matthew E Falagas Evridiki K Vouloumanou QU Jie-ming 《Chinese Medical Journal》 SCIE CAS CSCD 2013年第10期1965-1973,共9页
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 exami... 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. 展开更多
关键词 real-time polymerase chain reaction Pneumocystis pneumonia non-HIV immunocompromised patients HIV-positive
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Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice 被引量:5
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作者 FANG Yuan XU Li-hui TIAN Wen-xiao HUAI Yan YU Shan-hong LOU Miao-miao XIE Guan-lin 《Rice science》 SCIE 2009年第2期157-160,共4页
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 ... 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. 展开更多
关键词 Burkholderia glumae bacterial grain rot DETECTION real-time fluorescence polymerase chain reaction DCE
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Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus 被引量:6
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作者 Yuan Hui Zhiming Wu +12 位作者 Zhiran Qin Li Zhu Junhe Liang Xujuan Li Hanmin Fu Shiyu Feng Jianhai Yu Xiaoen He Weizhi Lu Weiwei Xiao Qinghua Wu Bao Zhang Wei Zhao 《Virologica Sinica》 SCIE CAS CSCD 2018年第3期270-277,共8页
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 esta... 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. 展开更多
关键词 Zika virus Nucleic acid detection - Micro-droplet digital polymerase chain reaction (ddPCR)real-time fluorescence quantitative polymerase chain reaction (RT-qPCR)
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Comparison of Abbott and Da-an real-time PCR for quantitating serum HBV DNA 被引量:2
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作者 Ning Qiu Rui Li +6 位作者 Jian-Guo Yu Wen Yang Wei Zhang Yong An Tong Li Xue-En Liu Hui Zhuang 《World Journal of Gastroenterology》 SCIE CAS 2014年第33期11762-11769,共8页
AIM: To compare the performance of the Da-an real-time hepatitis B virus (HBV) DNA assay and Abbott RealTime HBV assay.
关键词 Hepatitis B virus Hepatitis B virus DNA quantitation real-time polymerase chain reaction Chronic hepatitis B Antiviral therapy
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RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)
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作者 Yingyue ZHANG Jinxin YANG +2 位作者 Ze YANG Cong QI Di XU 《Journal of Oceanology and Limnology》 SCIE CAS CSCD 2023年第5期1910-1917,共8页
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.... 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. 展开更多
关键词 reference gene Gracilaria vermiculophylla life-history stage quantitative real-time polymerase chain reaction(RT-qPCR) red algae
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SMRT sequencing and ddPCR reveal the complexity of developmental trajectories and temporal dynamics of gut bifidobacterial communities in infants
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作者 Xu Gao Tao Zhang +5 位作者 Xiaoye Bai Qiannan Wen Dongyu Li Lai-Yu Kwok Heping Zhang Zhihong Sun 《Food Science and Human Wellness》 SCIE CSCD 2023年第5期1743-1750,共8页
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 studi... 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. 展开更多
关键词 INFANTS Gut microbiota BIFIDOBACTERIUM Diversity Single-molecule real-time(SMRT)sequencing Droplet digital polymerase chain reaction
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实时荧光定量PCR定量方法研究进展 被引量:17
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作者 廉红霞 高腾云 +2 位作者 傅彤 孙宇 李改英 《江西农业学报》 CAS 2010年第10期128-129,132,共3页
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。
关键词 实时荧光定量 PCR技术 定量方法 研究进展 polymerase chain reaction QUANTITATIVE real-time Method of 分子生物学研究 应用前景 特异性强 灵敏度高 封闭反应 重复性 速度 工具
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不同牙周状况龈下菌斑中福赛斯坦纳菌分布 被引量:5
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作者 杨禾 赵蕾 +4 位作者 孟姝 吴亚菲 付春华 赵寰 徐屹 《牙体牙髓牙周病学杂志》 CAS 2007年第8期444-447,共4页
目的:分析牙周健康者和慢性牙周炎病人龈下菌斑中福赛斯坦纳菌的分布情况,探讨该菌与不同牙周状况的关系。方法:收集111例牙周健康者、108例慢性牙周炎病人,病变位点和健康位点共327个龈下菌斑,采用16S rRNA PCR方法检测福赛斯坦纳菌的... 目的:分析牙周健康者和慢性牙周炎病人龈下菌斑中福赛斯坦纳菌的分布情况,探讨该菌与不同牙周状况的关系。方法:收集111例牙周健康者、108例慢性牙周炎病人,病变位点和健康位点共327个龈下菌斑,采用16S rRNA PCR方法检测福赛斯坦纳菌的分布。结果:牙周炎病人病变位点福赛斯坦纳菌检出率为56.5%,显著高于牙周健康者(10.8%,P<0.001)。慢性牙周炎病人健康位点福赛斯坦纳菌检出率为3.7%,显著低于病变位点(P<0.001),牙周健康者该菌的检出率比牙周炎健康位点高(P<0.05)。结论:龈下菌斑中福赛斯坦纳菌的存在与牙周炎症的发生有密切关系。 展开更多
关键词 牙周炎 福赛斯坦纳菌 聚合酶链反应
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不同牙周状况龈下菌斑中牙龈卟啉单胞菌和福赛斯坦纳菌的分布 被引量:4
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作者 杨禾 赵蕾 +3 位作者 孟姝 吴亚菲 赵寰 徐屹 《现代口腔医学杂志》 CAS CSCD 北大核心 2008年第1期20-24,共5页
目的分析中国牙周健康者和慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌和福赛斯坦纳菌的分布情况,探讨两种细菌在中国慢性牙周炎患者中的分布及其致病机理。方法收集65例牙周健康者、62例慢性牙周炎患者的龈下菌斑,采用16SrRNAPCR方法检测... 目的分析中国牙周健康者和慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌和福赛斯坦纳菌的分布情况,探讨两种细菌在中国慢性牙周炎患者中的分布及其致病机理。方法收集65例牙周健康者、62例慢性牙周炎患者的龈下菌斑,采用16SrRNAPCR方法检测牙龈卟啉单胞菌和福赛斯坦纳菌的分布。结果牙周健康者牙龈卟啉单胞菌和福赛斯坦纳菌的检出率分别是21.6%、12.3%,慢性牙周炎患者病变位点两菌检出率分别为74.2%、58.1%,牙周炎健康位点两菌的检出率分别是22.6%、4.8%。牙周炎病变位点两种细菌的检出率均明显高于牙周健康者和牙周炎健康位点(P<0.001);福赛斯坦纳菌在牙周健康者中的检出率高于牙周炎健康位点(P<0.05);牙周健康者和牙周炎健康位点牙龈卟啉单胞菌的检出率没有差异。慢性牙周炎患者两菌联合检出率为51.6%。结论牙龈卟啉单胞菌、福赛斯坦纳菌以及两种细菌联合感染与牙周炎密切相关。 展开更多
关键词 牙周炎 牙龈卟啉单胞菌 福赛斯坦纳菌 聚合酶链反应
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慢性牙周炎龈下菌斑中福赛斯坦纳菌的检测 被引量:2
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作者 杨禾 孟姝 +3 位作者 赵蕾 徐屹 吴亚菲 赵寰 《华西口腔医学杂志》 CAS CSCD 北大核心 2007年第5期454-457,共4页
目的检测慢性牙周炎患者和牙周健康者龈下菌斑中福赛斯坦纳菌的数量和所占比例,探讨福赛斯坦纳菌与牙周炎发生发展的关系。方法采集经常规聚合酶链反应(PCR)法检测福赛斯坦纳菌为阳性的61例慢性牙周炎患者和12例牙周健康者的龈下菌斑,应... 目的检测慢性牙周炎患者和牙周健康者龈下菌斑中福赛斯坦纳菌的数量和所占比例,探讨福赛斯坦纳菌与牙周炎发生发展的关系。方法采集经常规聚合酶链反应(PCR)法检测福赛斯坦纳菌为阳性的61例慢性牙周炎患者和12例牙周健康者的龈下菌斑,应用TaqMan实时荧光定量PCR法对菌斑的细菌总量和福赛斯坦纳菌的数量进行定量检测,构建含有福赛斯坦纳菌和真细菌靶基因的重组质粒,建立定量标准。结果本研究设计的引物和探针具有良好的特异性和敏感性;慢性牙周炎患者病变位点的福赛斯坦纳菌数量和细菌总量均高于牙周健康者的健康位点,且福赛斯坦纳菌在龈下菌斑中的比例也比健康位点高(P<0.05);龈下菌斑的细菌数量与探诊深度呈正相关(P<0.001);龈下菌斑中福赛斯坦纳菌所占比例在不同的探诊深度间无统计学差异(P>0.05)。结论龈下菌斑中福赛斯坦纳菌的数量与牙周状况有密切关系,实时荧光定量PCR法是研究牙周病病因及治疗方法的有效手段。 展开更多
关键词 牙周炎 实时荧光定量聚合酶链反应 福赛斯坦纳菌
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慢性牙周炎患者龈下菌斑中福赛斯坦纳菌与临床指标的关系 被引量:2
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作者 杨禾 吴亚菲 +1 位作者 赵蕾 孟姝 《华西口腔医学杂志》 CAS CSCD 北大核心 2007年第1期46-49,共4页
目的分析慢性牙周炎患者龈下菌斑中福赛斯坦纳菌(T.forsythensis)的分布情况,及其与牙周临床指标的关系,探讨T.forsythensis与慢性牙周炎发生发展的关系。方法采集108例慢性牙周炎患者的216个病变位点和108个健康位点的龈下菌斑,共324... 目的分析慢性牙周炎患者龈下菌斑中福赛斯坦纳菌(T.forsythensis)的分布情况,及其与牙周临床指标的关系,探讨T.forsythensis与慢性牙周炎发生发展的关系。方法采集108例慢性牙周炎患者的216个病变位点和108个健康位点的龈下菌斑,共324个样本,采用16SrRNA聚合酶链反应检测T.forsythensis的分布情况。对324个取样位点进行临床检查,记录探诊深度、临床附着丧失和探诊出血情况,分析T.forsythensis与临床指标的相互关系。结果慢性牙周炎患者病变位点与健康位点T.forsythensis的检出率分别为59.72%和3.70%,二者间有统计学差异(P<0.001)。随着探诊深度和临床附着丧失的增加,T.forsythensis的检出率也升高,Spearman相关系数分别为0.48和0.51。探诊出血阳性位点T.forsythensis的检出率为61.31%,明显高于探诊出血阴性位点的检出率8.80%(P<0.001)。结论T.forsythensis与慢性牙周炎发生、发展关系密切。 展开更多
关键词 牙周炎 福赛斯坦纳菌 聚合酶链反应
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Update on occult hepatitis B virus infection 被引量:33
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作者 Manoochehr Makvandi 《World Journal of Gastroenterology》 SCIE CAS 2016年第39期8720-8734,共15页
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... 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. 展开更多
关键词 Nested polymerase chain reaction Occult hepatitis B infection CRYPTOGENIC real-time polymerase chain reaction
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