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A sensitive SYBR Green rt-qpcr method for grapevine virus E and its application for virus detection in different grapevine sample types

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Journal of Integrative Agriculture 2020年第7期19卷 1834-1841页

作者： REN Fang ZHANG Zun-ping FAN Xu-dong HU Guo-jun ZHANG Meng-yan DONG Ya-feng National Center for Eliminating Viruses from Deciduous Fruit Trees Research Institute of PomologyChinese Academy of Agricultural SciencesXingcheng 125100P.R.China

To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE),a SYBR Green based real-time fluorescence quantitative rt-PCR method(rt-qpcr)was *** method could be used to detect GVE specifically,and the sensitivity was about 100 times greater than conventional *** excellent linear correlation(R=0.997)and a high amplification efficiency(E=97.5%)were obtained from the standard curve of this *** tests revealed that the coefficients of variation in the intra-and inter-assay results were 0.31-1.03%and 0.82--262%,respectively,indicating a good *** rt-qpcr method could be used to detect GVE in a wide range of grapevine sample *** detection rates of rt-qpcr for nearly all sample types from different positions and seasons were higher than conventional *** detection rates in spring,summer,autumn and winter increased *** in autumn and winter were best for detection,and the detection rates of most samples were 80-100%,which were 10 to 40%higher than conventional *** general,old petioles and branches were the best tissues for GVE *** detection rates of these samples in each season were all 100%,which were 20 to 40%higher than conventional *** second highest rates were in the old leaf,with detection rates for rt-qpcr of 80-100%in all seasons,which were 20 to 40%higher than conventional *** could be difficultly detected in young leaves by conventional rt-PCR,and the detection rates were only 0-50%,while by rt-qpcr the rates could increase to 0--80%.A total of 33 out of 363 samples(belonging to 68 cultivars)from 20 regions in China were detected to be positive by rt-qpcr(9.1%),which was more than twice the rate of the conventional rt-PCR(3.9%).

关键词： grapevine grapevine virus E detection rt-qpcr conventional rt-PCR

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Influence of storage solution, temperature, assay time and concentration on rt-qpcr nucleic acid detection for SARS-CoV-2 detection of SARS-CoV-2 by the rt-qpcr

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2024年 707卷 149726页

作者： Su, Lingxuan Lou, Yihan Li, Jiaxuan Mao, Haiyan Li, Jianhua Sun, Yi Zhou, Biaofeng Wu, Guangshang Huang, Chen Zhang, Yanjun Chen, Keda Zhejiang Prov Ctr Dis Control & Prevent Hangzhou Peoples R China Zhejiang Shuren Univ Shulan Int Med Coll Key Lab Articial Organs & Computat Med Zhejiang Pr Hangzhou Peoples R China Key Lab Publ Hlth Detect & Etiol Res Zhejiang Prov Hangzhou Peoples R China

Real-time reverse transcriptase quantitative polymerase chain reaction (rt-qpcr) is an important method for the early diagnosis of coronavirus disease 2019 (COVID-19). This study investigated the effects of storage solution, temperature and detection time on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection by rt-qpcr. Various concentrations of SARS-CoV-2 were added to inactive and non-inactive storage solution and the viral suspensions were stored at various temperatures (room temperature, 4, -20 and -80 degrees C). Then, at five different detection time points, the Ct values were determined by rt-qpcr. Active and inactive storage solutions and storage temperature have a great impact on the detection of N gene of SARS-CoV-2 at different concentration corridors but have little impact on the ORF gene. The storage time has a greater impact on the N gene and ORF gene at high concentrations but has no effect on the two genes at low concentrations. In conclusion, storage temperature, storage time and storage status (inactivated, non-inactivated) have no effect on the nucleic acid detection of SARS-CoV-2 at the same concentration. For different concentrations of SARS-CoV-2, the detection of N gene is mainly affected.

关键词： rt-qpcr SARS-CoV-2 Viral storage solution Storage temperature Detection time

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Development of rt-qpcr and loop-mediated isothermal amplification (LAMP) assays for the rapid detection of Rahnella aquatilis in fish

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AQUACULTURE 2024年 578卷

作者： Shi, Meijuan Cao, Yanyan Hu, Xiucai Luo, Fuli Lv, Aijun Tianjin Agr Univ Coll Fisheries Tianjin Key Lab Aqua Ecol & Aquaculture Tianjin 300384 Peoples R China

Rahnella aquatilis is widely recognized as an opportunistic pathogen of fish, but molecular detection and diagnostic techniques for it remain to be developed. In a previous study, we isolated the pathogenic R. aquatilis strain KCL-5 from diseased crucian carp Carassius auratus and identified it as a new fish pathogen causing enteritis and septicemia. Here, we describe novel real-time quantitative PCR (rt-qpcr) and loop-mediated isothermal amplification (LAMP) assays for the rapid and sensitive detection of R. aquatilis. Specific rt-qpcr and LAMP primers were developed for the outer membrane protein (OmpA) gene. Their reaction systems were optimized using the recombinant plasmid pMD18-T containing the OmpA gene and their specificities were confirmed using various bacterial fish pathogens including Aeromonas hydrophila, A. veronii, A. allosaccharophila, Yersinia ruckeri, Vibrio vulnificus, V. alfacsensis, Providencia rettgeri, and R. aquatilis. The sensitivities of the rt-qpcr and LAMP assays were further evaluated using serial dilutions of the positive recombinant plasmid pMD18-T/OmpA. Both the rt-qpcr and LAMP assays were able to reliably detect the presence of R. aquatilis with high accuracy and a specificity of 100%. The lowest detection limits of the rt-qpcr and LAMP assays were 2.3 copies/mu L and 2.3 x 101 copies/mu L, respectively. To the best of our knowledge, this is the first report of rt-qpcr and LAMP methods for R. aquatilis detection, and provides a means for its rapid detection and diagnosis in fish.

关键词： Rahnella aquatilis rt-qpcr LAMP Diagnosis Fish

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Selection of appropriate reference genes for rt-qpcr analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs

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GENE 2015年第1期558卷 152-158页

作者： Park, Sang-Je Kwon, Seul Gi Hwang, Jung Hye Park, Da Hye Kim, Tae Wan Kim, Chul Wook Korea Res Inst Biosci & Biotechnol Natl Primate Res Ctr Chungbuk 363883 South Korea Gyeongnam Natl Univ Sci & Technol Swine Sci & Technol Ctr Jinju 660758 South Korea

Reverse transcription quantitative real-time polymerase chain reaction (rt-qpcr) is the most reliable molecular biology technique for assessment of mRNA expression levels. However, to obtain the accurate rt-qpcr results, the expression levels of genes of interest should be normalized with appropriate reference genes and optimal numbers of reference genes. In this study, we assessed the expression stability of 15 well-known candidate reference genes (ACTB, ALDOA, B2M, GAPDH, HPAR1, HSPCB, PGK1, POLR2G, PPIA, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ) in seven body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire, Landrace, Duroc, and Yorkshire pigs using three excel-based programs, geNorm, NormFinder, and BestKeeper. Combination analysis of these three programs showed that the stable and appropriate reference genes are PPIA, TBP, and HSPCB in Berkshire pigs;PPIA, TBP, RPL4, and RPS18 in Landrace pigs;PPIA and TBP in Duroc pigs;and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs. Because the four pig breeds had different suitable reference genes, the selection of appropriate reference genes is essential in rt-qpcr analyses. Taken together, our data could help to select reliable reference genes for the normalization of expression levels of various target genes in pigs. (C) 2015 Elsevier B.V. All rights reserved.

关键词： Berkshire Duroc Landrace Normalization Reference gene rt-qpcr Yorkshire

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A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for rt-qpcr

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APMIS 2021年第7期129卷 393-400页

作者： Kalnina, Lelde Mateu-Regue, Angels Oerum, Stephanie Hald, Annemette Gerstoft, Jan Oerum, Henrik Nielsen, Finn Cilius Iversen, Astrid K. N. Rigshosp Ctr Genom Med Copenhagen Denmark Univ Paris Diderot Inst Physicochem Biol IBPC CNRS Paris France Natl Univ Hosp Dept Infect Dis Rigshosp Copenhagen Denmark CIVI Biopharma Inc Washington DC USA Univ Oxford John Radcliffe Hosp Div Clin Neurol Nuffield Dept Clin NeurosciWeatherall Inst Mol M Oxford England

The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step rt-qpcr for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step rt-qpcr against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by rt-qpcr with high sensitivity (similar to 4 viral RNA copies per rt-qpcr). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in rt-qpcr detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.

关键词： SARS-CoV-2 RNA extraction virus inactivation SARS-CoV-2 rt-qpcr clinical microbiology molecular microbiology rapid diagnostic methods rt-qpcr virology

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Development and validation of a TaqMan rt-qpcr for the detection of convert mortality nodavirus (CMNV)

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JOURNAL OF VIROLOGICAL METHODS 2018年 262卷 65-71页

作者： Li, Xiao-Ping Wan, Xiao-Yuan Xu, Ting-Ting Huang, Jie Zhang, Qing-Li Chinese Acad Fishery Sci Yellow Sea Fisheries Res InstQingdao Natl Lab Ma Qingdao Key Lab Mariculture Epidemiol & Biosecur Key Lab Maricultural Organ Dis ControlMinist Agr Qingdao 266071 Peoples R China Shanghai Ocean Univ Natl Demonstrat Ctr Expt Fisheries Sci Educ Shanghai 201306 Peoples R China

Covert mortality nodavirus (CMNV), an emerging RNA virus, is the pathogen of viral covert mortality disease (VCMD), which has emerged as a cause of serious losses in shrimp aquaculture in China. To improve VCMD diagnosis, a one-step, real-time TaqMan probe-based reverse transcription quantitative PCR (rt-qpcr) was developed in this study. The TaqMan rt-qpcr was optimized firstly, whereby the best results were obtained with 0.2 mu M of each primer, 0.2 mu M probe, and 0.5 mu L Enzyme Mix II. The optimal reaction program was determined as 15 min at 51 degrees C for reverse transcription and 5 min at 95 degrees C, followed by 40 cycles of denaturation at 94 degrees C for 10 s, and annealing and extension at 52.7 degrees C for 30 s. The optimized assay detected as little as 9.6 pg total RNA from CMNV-infected shrimp and 5.7 copies of the target plasmid. The rt-qpcr assay for CMNV with a high correlation coefficient (r(2) = 0.996) was developed basing on the standard curve generated by plotting the threshold cycle values (y) against the common logarithmic copies (log(10)n(c) as x;n(c) is copy number) of pMD20-CMNV. The diagnostic sensitivity and specificity of this assay versus the previously reported rt-qpcr was 96.2% and 98.0%, respectively. This method is highly specific to CMNV, as it showed no cross-reactivity with other common shrimp viruses. It is anticipated that the newly developed and optimized rt-qpcr assay will be instrumental for the rapid diagnosis and quantitation of CMNV.

关键词： Covert mortality nodavirus CMNV TaqMan rt-qpcr

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Development and validation of an rt-qpcr assay for rapid detection and quantification of hepatitis C virus RNA for routine testing in Moroccan clinical specimens

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JOURNAL OF MEDICAL VIROLOGY 2019年第3期91卷 428-436页

作者： El Hadi, Hicham Benani, Abdelouaheb Qmichou, Zineb Lazar, Fatiha Bakri, Youssef Benhassou, Hassan Ait Moumen, Abdeladim Moroccan Fdn Adv Sci Innovat & Res MASCIR Ctr Med Biotechnol Div Biotechnol Rabat Morocco Mohamed V Univ Fac Sci Biol Human Pathol Lab Rabat Morocco Pasteur Inst Morocco Mol Biol Lab Casablanca Morocco

A one-step reverse transcription quantitative PCR (rt-qpcr) assay in combination with rapid RNA extraction was evaluated for routine testing of hepatitis C virus (HCV) RNA. Specific primers and probes were designed for the detection of a 150 bp sequence located in the 5 ' untranslated region (5 ' UTR) of HCV RNA. The target sequence was selected as the most conserved region between the six known HCV subtype sequences following an alignment. The assay was able to quantify a dynamic linear range of 10(8) to 10(1) plasmid copies/reaction (r(2) = 0.98) containing the target sequence. Two copies of this HCV plasmid corresponds to one international unit (IU) measured using a standard obtained by serial dilutions of the World Health Organization (WHO) standard. The detection limit of the assay was about 10 IU/mL of HCV RNA (20 copies/mL) in plasma samples. The assay was comparable to Cobas AmpliPrep/Cobas TaqMan (R) HCV Test, v2.0 Quantitative assay (Roche Molecular Systems, Inc., Branchburg, NJ) with correlation coefficient r(2) = 0.98. The present assay could be completed within 3 hours from RNA extraction to data analysis of at least 30 plasma samples. Our test provides sufficient sensitivity, specificity, and reproducibility and proved to be fast, labor-saving, and cost-effective. Indeed, our system will definitely allow low-income countries to monitor accurately this viral infection and to efficiently treat their infected patients.

关键词： Hepatitis C molecular diagnostic rt-qpcr viral load quantification

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Reference gene selection and validation for mRNA expression analysis by rt-qpcr in murine M1-and M2-polarized macrophage

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MOLECULAR BIOLOGY REPOrtS 2020年第4期47卷 2735-2748页

作者： Ju, Wen Sun, Tiantian Lu, Wenyi Smith, Alhaji Osman Bao, Yurong Adzraku, Seyram Yao Qi, Kunming Xu, Kailin Qiao, Jianlin Zeng, Lingyu Xuzhou Med Univ Blood Dis Inst Xuzhou 221002 Jiangsu Peoples R China Key Lab Bone Marrow Stem Cell Xuzhou 221002 Jiangsu Peoples R China Xuzhou Med Univ Affiliated Hosp Dept Hematol Xuzhou 221002 Jiangsu Peoples R China

Murine bone marrow-derived macrophages (M0) and M1- and M2-polarized macrophages are being widely used as a laboratory model for polarized macrophages related molecular mechanism analysis. Gene expression analysis based on reference gene normalization using rt-qpcr was a powerful way to explore the molecular mechanism. But little is known about reference genes in these cell models. So, the goal of this study was to identify reference genes in these types of macrophages. Candidate reference genes in murine bone marrow-derived and polarized macrophages were selected from microarray data using Limma linear model method and evaluated by determining the stability value using five algorithms: BestKeeper, NormFinder, GeNorm, Delta CT method, and RefFinder. Finally, the selected stable reference genes were validated by testing three important immune and inflammatory genes (NLRP1, IL-1 beta, and TNF-alpha) in the cell lines. Our study has clearly shown that Ubc followed by Eef1a1 and B2m respectively were recognized as the three ideal reference genes for gene expression analysis in murine bone marrow-derived and polarized macrophages. When three reference genes with strong different stability were used for validation, a large variation of a gene expression level of IL-1 beta, TNF-alpha and NLRP1 were obtained which provides clear evidence of the need for careful selection of reference genes for rt-qpcr analysis. Normalization of mRNA expression level with Ubc rather than Actb or Gusb by qpcr in macrophages and polarized macrophages is required to ensure the accuracy of the qpcr analysis.

关键词： Reference gene rt-qpcr Macrophages Polarization

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mRNA and microRNA quality control for rt-qpcr analysis

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METHODS 2010年第4期50卷 237-243页

作者： Becker, C. Hammerle-Fickinger, A. Riedmaier, I. Pfaffl, M. W. Tech Univ Munich Chair Physiol D-85354 Freising Weihenstephan Germany

The importance of high quality sample material, i.e. non-degraded or fragmented RNA, for classical gene expression profiling is well documented. Hence, the analysis of RNA quality is a valuable tool in the preparation of methods like rt-qpcr and microarray analysis. For verification of RNA integrity, today the use of automated capillary electrophoresis is state of the art. Following the recently published MIQE guidelines, these pre-PCR evaluations have to be clearly documented in scientific publication to increase experimental transparency. RNA quality control may also be integrated in the routine analysis of new applications like the investigation of microRNA (miRNA) expression, as there is little known yet about factors compromising the miRNA analysis. Agilent Technologies is offering a new lab-on-chip application for the 2100 Bioanalyzer making it possible to quantify miRNA in absolute amounts [pg] and as a percentage of small RNA [%]. Recent results showed that this analysis method is strongly influenced by total RNA integrity. Ongoing RNA degradation is accompanied by the formation of small RNA fragments leading to an overestimation of miRNA amount on the chip. Total RNA integrity is known to affect the performance of rt-qpcr as well as the quantitative results in mRNA expression profiling. The actual study identified a comparable effect for miRNA gene expression profiling. Using a suitable normalization method could partly reduce the impairing effect of total RNA integrity. (C) 2010 Elsevier Inc. All rights reserved.

关键词： miRNA mRNA RNA quality Gene expression rt-qpcr Normalization

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Short-term response of different Saccharomyces cerevisiae strains to hyperosmotic stress caused by inoculation in grape must: rt-qpcr study and metabolite analysis

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FOOD MICROBIOLOGY 2015年 52卷 49-58页

作者： Noti, Olta Vaudano, Enrico Pessione, Enrica Garcia-Moruno, Emilia Consiglio Ric Agr & Anal Econ Agr Ctr Ric Enol I-14100 Asti Italy Univ Turin Dipartimento Sci Vita & Biol Sistemi I-10123 Turin Italy

During the winemaking process, glycerol synthesis represents the first adaption response of Saccharomyces cerevisiae to osmotic stress after inoculation in grape must. We have implemented an rt-qpcr (Reverse Transcription-quantitative PCR) methodology with a preventive evaluation of candidate reference genes, to study six target genes related to glycerol synthesis (GPD1, GPD2, GPP2 and GPP1) and flux (STL1 and FPS1), and three ALD genes coding for aldehyde dehydrogenase involved in redox equilibrium via acetate production. The mRNA level in three strains, characterized by different metabolite production, was monitored in the first 120 min from inoculation into natural grape must. Expression analysis shows a transient response of genes GPD1, GPD2, GPP2, GPP1 and STL1 with differences among strains in term of mRNA abundance, while FPS1 was expressed constitutively. The transient response and different expression intensity among strains, in relation to the intracellular glycerol accumulation pattern, prove the negative feedback control via the HOG (High Osmolarity Glycerol) signalling pathway in S. cerevisiae wine strains under winery conditions. Among the ALD genes, only ALD6 was moderately induced in the hyperosmotic environment but not in all strains tested, while ALD3 and ALD4 were drastically glucose repressed. The intensity of transcription of ALD6 and ALD3 seems to be related to different acetate production found among the strains. (C) 2015 Elsevier Ltd. All rights reserved.

关键词： Saccharomyces cerevisiae Glycerol rt-qpcr Hyperosmosis Wine

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