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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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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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Quantitative studies of the regular distribution pattern for Salmonella enteritidis in the internal organs of mice after oral challenge by a specific real-time polymerase chain reaction 被引量:7
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作者 Shu-Xuan Deng An-Chun Cheng +5 位作者 Ming-Shu Wang Ping Cao Bin Yan Nian-Chun Yin Sheng-Yan Cao Zhen-Hua Zhang 《World Journal of Gastroenterology》 SCIE CAS CSCD 2008年第5期782-789,共8页
AIM: To identify and understand the regular distribution pattern for Salmonella enteritidis (S. enteritidis) in the internal organs of mice after an oral challenge over a 3 wk period. METHODS: Assays based on the ... AIM: To identify and understand the regular distribution pattern for Salmonella enteritidis (S. enteritidis) in the internal organs of mice after an oral challenge over a 3 wk period. METHODS: Assays based on the serovar-specific DNA sequence of S. enteritidis from GenBank, and a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) were developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the blood and the internal organs, including heart, liver, spleen, kidney, pancreas, and gallbladder, from mice after oral challenge at different time points respectively.RESULTS: The results showed that the spleen was positive at 12 h post inoculation (PI), and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas was positive at 20 h PI, and the final organs to show positive results were the kidney and gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h PI, with the liver and spleen containing high concentrations of S. enteritidis, whereas the blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 12 d PI in the gallbladder, 2 wk for the liver, and 3 wk for the spleen without causing apparent symptoms.CONCLUSION: The results provided significant data for understanding the life cycle of S. enteritidis in the internal organs, and showed that the liver and spleen may be the primary sites for setting itself up as a commensa over a long time after oral challenge. Interestingly, it may be the first time reported that the gallbladder is a site of carriage for S. enteritidis over a 12 d period. This study will help to understand the mechanisms of action of S. enteriCdis infection in vivo. 展开更多
关键词 Fluorescence-based quantitative polymerase chain reaction Internal organs Salmonella enteritidis Regular distribution pattern
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Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay 被引量:2
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作者 Zhu Yang Guoliang Mao +8 位作者 Yujun Yuan-Chuan Chen Chengjing Liu Jun Luo Xihan Li Ke Zen Yanjun Pang Jianguo Wu Fenyong Liu 《Virologica Sinica》 SCIE CAS CSCD 2013年第1期24-35,共12页
A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and... A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection. 展开更多
关键词 quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) Influenza A virus DETECTION
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Confusing finding of quantitative fluorescent polymerase chain reaction analysis in invasive prenatal genetic diagnosis:A case report
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作者 Cui Chen Tao Tang +2 位作者 Qi-Ling Song Yong-Jun He Yan Cai 《World Journal of Clinical Cases》 SCIE 2023年第28期6895-6901,共7页
BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in di... BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing. 展开更多
关键词 quantitative fluorescent polymerase chain reaction Copy number variation sequencing Prenatal diagnosis Partial duplication KARYOTYPING Case report
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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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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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Gastrointestinal tract distribution of Salmonella enteritidis in orally in fected mice with a species-specific fluorescent quantitative polymerase chain reaction 被引量:12
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作者 Shu-Xuan Deng An-Chun Cheng +1 位作者 Ming-Shu Wang Ping Cao 《World Journal of Gastroenterology》 SCIE CAS CSCD 2007年第48期6568-6574,共7页
AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA seque... AIM: To identify and understand the regular distribution pattern and primary penetration site for Salmonella enteritidis (S. enteritidis) in the gastrointestinal tract. METHODS: Based on the species-specific DNA sequence of S. enteritidis from GenBank, a species-specific real- time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) was developed for the detection of S. enteritidis. We used this assay to detect genomic DNA of S. enteritidis in the gastrointestinal tract, including duodenum, jejunum, ileum, cecum, colon, rectum, esophagus and stomach, from mice after oral infection. RESULTS: S. enteritidis was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive at 8 h post inoculation, and the final organ to show a positive result was the stomach at 18 h post inoculation. The copy number of S. enteritidis DNA in each tissue reached a peak at 24-36 h post inoculation, with the jejunum, ileum and cecum containing high concentrations of S. enteritidis, whereas the duodenum, colon, rectum, stomach and esophagus had low concentrations. S. enteritidis began to decrease and vanished at 2 d post inoculation, but it was still present up to 5 d post inoculation in the jejunum, ileum andcecum, without causing apparent symptoms. By 5 d post inoculation, the cecum had significantly higher numbers of S. enteritidis than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for S. enteritidis. CONCLUSION: The results provided significant data for clarifying the pathogenic mechanism of S. enteritidis in the gastrointestinal tract, and showed that the jejunum, ileum and cecum are the primary sites of invasion in normal mice after oral infection. This study will help to further understanding of the mechanisms of action of S. enteritidis. 展开更多
关键词 Fluorescence-based quantitative polymerase chain reaction Gastrointestinal tract Salmonella enteritidis
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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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A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao 被引量:1
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作者 GUO Hao LIU Yongjian +3 位作者 ZHANG Qi YUAN Xiutang ZHANG Weiwei ZHANG Zhifeng 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2015年第2期132-136,共5页
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
关键词 Aureococcus anophagefferens quantitative polymerase chain reaction(q PCR) field samples
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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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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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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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Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application 被引量:5
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作者 Zhao, Wei-Feng Shao, You-Lin +4 位作者 Chen, Liang-Yun Wu, Jin-Hua Zhu, Yi-Ling Gan, Jian-He Xiong, Hui 《World Journal of Gastroenterology》 SCIE CAS CSCD 2010年第10期1267-1273,共7页
AIM:To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies.METHODS: Based on the characteristics of rtA181V/T and... AIM:To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies.METHODS: Based on the characteristics of rtA181V/T and rtN236T mutations, a new approach based on real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was established for the detection of ADV-resistant HBV quasispecies, total HBV DNA, rtA181 and rtN236 mutations in blood samples from 32 chronic hepatitis B (CHB) patients with unsatisfactory curative effect on ADV and compared with routine HBV DNA sequencing.RESULTS: Both the sensitivity and specificity of this new detection approach to ADV-resistant HBV quasispecies were 100%, which were much higher than those of direct HBV DNA sequencing. The approach was able to detect 0.1% of mutated strains in a total plasmid population. Among the 32 clinical patients, single rtA181 and rtN236T mutation and double rtA181T and rtN236T mutations were detected in 20 and 8, respectively, while ADV-resistant mutations in 6 (including, rtA181V/T mutation alone in 5 patients) and no associated mutations in 26.CONCLUSION: This new approach is more feasible and efficient to detect ADV-resistant mutants of HBV and ADV-resistant mutations before and during ADV treatment with a specificity of 100% and a sensitivity of 100%. 展开更多
关键词 Chronic hepatitis B ADEFOVIR Drug resistance quantitative detection real-time fluorescent quantitative polymerase chain reaction
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Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR 被引量:9
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作者 Anastasia Pavlidou Maria Dalamaga +4 位作者 Christos Kroupis George Konstantoudakis Maria Belimezi George Athanasas Kleanthi Dimas 《World Journal of Gastroenterology》 SCIE CAS CSCD 2011年第12期1614-1621,共8页
AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and... AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore(LC-Red 640).Real time quantitative polymerase chain reaction(PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues.In the patients group,carcinoembryonic antigen(CEA) and CA19-9 tumor markers were also measured immunochemically.RESULTS:Wild type survivin mRNA isoform was expressed in 48%of the 52 tumor samples,survivin-2b in 38%and survivin-ΔΕx3 in 29%,while no expression was found in normal tissues.The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b,survivin-ΔΕx3,survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin(P<0.001).The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion(P=0.006 and P<0.005,respectively).A significant difference was found between survivin-2b and morphologic cancer type.Also,the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis.No association was observed between the three isoforms and grade,metastasis,Dukes stage and gender.The three isoforms were not correlated with CEA and CA19-9.CONCLUSION:Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer. 展开更多
关键词 SURVIVIN mRNA isoforms Apoptosis gene Colorectal adenocarcinomas Real time quantitative polymerase chain reaction LIGHTCYCLER
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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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Quantitative Analysis of ATP Sulfurylase and Selenocysteine Methyltransferase Gene Expression in Different Organs of Tea Plant (<i>Camellia sinensis</i>) 被引量:3
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作者 Shaoqiang Tao Juan Li +4 位作者 Xungang Gu Yanan Wang Qiang Xia Bing Qin Lin Zhu 《American Journal of Plant Sciences》 2012年第1期51-59,共9页
Tea plant (Camellia sinensis) has unique biological features for the study of cellular and molecular mechanisms, an evergreen broad-leaved woody plant which can accumulate selenium in soil abundant of Selenium. Expres... Tea plant (Camellia sinensis) has unique biological features for the study of cellular and molecular mechanisms, an evergreen broad-leaved woody plant which can accumulate selenium in soil abundant of Selenium. Expression of the genes related to Selenium (Se) metabolism is an adaptation to the soil environment for a long period. The purpose of the present study was to explore if there exist differences of expression about these genes in tea plant between growing in Selenium-abundant and normal soil. A quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR) assay was done for quantification of ATP sulfurylase (APS) and selenocysteine methyltransferase (SMT) mRNA normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in tea plant. Young leaves, mature leaves and tender roots from tea plants growing in soil abundant of Selenium were respectively obtained from Shitai County, Anhui Province, and also the relevant materials of the selenium un-enriched tea plant planted at agricultural garden of Ahui Agriculture University were taken as control for real-time PCR analysis. The results showed that APS1, APS2 and SMT expression levels for either young or mature leaves in selenium-enriched tea plant were lower than that in ordinary (selenium un-enriched) tea plant. In contrast, the APS1, APS2 and SMT expression level of roots in selenium-enriched tea plant were all higher than that in ordinary tea plant. APS1 gene expression level of roots in selenium-enriched tea plant was about 1.6 times higher than that in the ordinary tea plant, APS2 gene expression level was about 4.8-fold higher than that in the ordinary tea plant, SMT gene expression level was about 3.3 times higher than that in the ordinary tea plant. Among various tissues of selenium-enriched tea plant, APS1 gene relative expression level of young leaves was similar to or slightly higher than mature leaves, and the one of roots was the lowest among them;APS2 gene relative expression level of young leaves was similar to or slightly higher than the roots, and the one of mature leaves was the lowest among them;SMT gene relative expression level of young leaves was similar to or slightly higher than mature leaves, and the one of roots was the highest among them. Our results suggest that there existed correlation between selenium and expression levels of these genes. 展开更多
关键词 quantitative real-time polymerase chain reaction ATP Sulfurylase SELENOCYSTEINE METHYLTRANSFERASE Tea Plant (Camellia sinensis)
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