Latent Infection of Powdery Mildew on Volunteer Wheat in Sichuan Province, China

Wheat powdery mildew, caused by the fungal pathogen Blumeria graminis f. sp. tritici, is one of the most destructive wheat diseases in China, especially in Sichuan Province. Successfully oversummered B. graminis f. sp. tritici can become a primary infection source for wheat seedlings in the fall. Determining the latent infection level of B. graminis f. sp. tritici in volunteer wheat and the oversummering areas of B. graminis f. sp. tritici is important for estimating potential B. graminis f. sp. tritici epidemics. In this study, we clarified the critical role of volunteer wheat in the B. graminis f. sp. tritici oversummering cycle and determined whether latent B. graminis f. sp. tritici infection was present in volunteer wheat by using real-time polymerase chain reaction (real-time PCR). The results indicated that volunteer wheat was mostly found in the northeast and middle regions of Sichuan, where lower temperatures and higher precipitation are common. A total of 13.2% of samples showed symptoms of B. graminis f. sp. tritici (spores) in the field, and 36.8% of samples were found to carry the B. graminis f. sp. tritici pathogen, even though no symptoms were observed. Volunteer wheat with B. graminis f. sp. tritici infection symptoms was found at an altitude of 536 m but volunteer wheat latently infected by B. graminis f. sp. tritici was identified at the lowest altitude of 323 m. Crop shade (e.g., corn and lima bean) provided suitable conditions for the survival of volunteer wheat in the summer. In addition, volunteer wheat played a key role in the B. graminis f. sp. tritici oversummering cycle. Moreover, B. graminis f. sp. tritici could oversummer by infecting generations of volunteer wheat in the summer, thereby becoming the primary infection source for autumn-sown wheat. The results showed that the latent infection of wheat diseases could be rapidly quantified by real-time PCR. Here, the primary disease center of autumn-sown wheat in Ya’an and Wenjiang were detected accurately based on this method. This study provides solid evidence for identifying the disease center, which offers guidance for wheat disease control and management.

Download Full-text

Development of a LUX real-time PCR for the detection and quantification of human herpesvirus 7

Canadian Journal of Microbiology ◽

10.1139/w08-134 ◽

2009 ◽

Vol 55 (3) ◽

pp. 319-325 ◽

Cited By ~ 5

Author(s):

Massimiliano Bergallo ◽

Cristina Costa ◽

Maria Elena Terlizzi ◽

Francesca Sidoti ◽

Samuela Margio ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Primary Infection ◽

Latent Infection ◽

Dynamic Range ◽

Human Herpesvirus ◽

Clinical Samples ◽

House Light ◽

Pcr Assay ◽

Sensitivity Specificity

Human herpesvirus 7 is a highly seroprevalent β-herpesvirus that, following primary infection, remains latent in CD4+ T cells and determines a persistent rather than a latent infection in various tissues and organs, including the lung and skin. This paper describes the development of an in-house light upon extension real-time PCR assay for quantification of human herpesvirus 7 DNA in clinical samples. The efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated by evaluating the human herpesvirus 7 load in bronchoalveolar lavages and skin specimens, chosen as 2 persistency sites, from healthy and pathological individuals. The real-time PCR assay developed in this study could be a useful tool to detect and quantify human herpesvirus 7 DNA in different clinical specimens to elucidate its epidemiological and pathogenic roles.

Download Full-text

Real-time PCR quantification of latent infection of wheat powdery mildew in the field

European Journal of Plant Pathology ◽

10.1007/s10658-013-0188-5 ◽

2013 ◽

Vol 136 (3) ◽

pp. 565-575 ◽

Cited By ~ 8

Author(s):

Yaming Zheng ◽

Yong Luo ◽

Yilin Zhou ◽

Xiaowei Zeng ◽

Xiayu Duan ◽

...

Keyword(s):

Powdery Mildew ◽

Real Time ◽

Real Time Pcr ◽

Latent Infection ◽

Wheat Powdery Mildew

Download Full-text

Novel Role of the TAL1/SCL Transcription Factor in Murine Monocytopoiesis.

Blood ◽

10.1182/blood.v112.11.1376.1376 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1376-1376

Author(s):

Soumyadeep Dey ◽

David J. Curtis ◽

Stephen Jane ◽

Stephen J. Brandt

Keyword(s):

Transcription Factor ◽

Real Time ◽

Real Time Pcr ◽

Mononuclear Cells ◽

Critical Role ◽

Pcr Analysis ◽

Bhlh Transcription Factor ◽

Lacz Gene ◽

Macrophage Differentiation ◽

In Cells

Abstract The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-ã-activated macrophages. To elucidate Tal1’sfunctions in murine monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1fl/fl/lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1-null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1,PU.1, Csfr1, Msr1 (mouse scavenger receptor), Cd68, or Il6ra. Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining ofTal1-null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.

Download Full-text

Identification of epigenetic silencing of GCNT2 expression by comprehensive real-time PCR screening in colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.3_suppl.506 ◽

2014 ◽

Vol 32 (3_suppl) ◽

pp. 506-506

Author(s):

Kazunorii Nakamura ◽

Horomichi Sawaki ◽

Keishi Yamashita ◽

Masahiko Watanabe ◽

Hisashi Narimatsu

Keyword(s):

Colorectal Cancer ◽

Dna Methylation ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Critical Role ◽

Normal Mucosa ◽

Primary Cancer ◽

Normal Tissues ◽

Cancer Tissues

506 Background: Glycoprotein expression profile has been proved to be dramatically altered in human cancers, however specific glycogenes which are aberrant in expression in cancer cells has not been fully identified. Recent accumulated evidence supported notion that the reduced expression of tumor suppressor genes is explained by DNA promoter methylation in human cancer. Methods: We used Comprehensive Real time PCR system (CRPS) for glycogenes (189 genes) to identify genes aberrantly expressed in colorectal cancer tissues (CRC) as compared to the corresponding normal mucosa tissues. GCNT2 was of particular interest among the identified genes in CRC. Results: (1) GCNT2 harbors 3 isoforms which have different promoter regions. (2) All of the 3 isoforms of GCNT2 genes were remarkably decreased in CRC as compared to the corresponding normal mucosa, and each isoform expression was strongly associated with other 2 isoforms in primary cancer tissues by TaqMan real time PCR (R = 0.99-995, p < 0.0001). (3) Among the 5 CRC cell lines (DLD1, HCT116, CACO2, LOVO), those which were silenced in expression were reactivated by demethylating agents such as 5-aza-2’ deoxycytidine and trichostatin A. (4) Promoter region of the variant 2 of GCNT2 was consistent with its silenced expression in CRC cell lines by cloned sequence, so we examined DNA methylation status of the promoter of the GCNT2 variant 2 in 50 primary cancer tissues and the corresponding normal tissues. Quantitative MSP revealed that almost half of normal tissues have methylation as high as tumor tissues, while, in the primary CRC with less methylation in the corresponding normal tissues, DNA methylation was higher in primary CRC tissues than in the corresponding normal tissues. Finally, GCNT2 variant 2 stable transfection induced expression of other 2 isoform variants. Conclusions: We identified novel methylation gene GCNT2 among the glycoenes. Glycoenes that were altered in genomic or epigenetic manner have been few, so GCNT2 may play a critical role in cancer progression through glycan change.

Download Full-text

A new method for early detection of latent infection by ‘Candidatus Liberibacter asiaticus’ in citrus trees

F1000Research ◽

10.12688/f1000research.51546.1 ◽

2021 ◽

Vol 10 ◽

pp. 250

Author(s):

Kazuki Fujiwara ◽

Kenta Tomimura ◽

Toru Iwanami ◽

Takashi Fujikawa

Keyword(s):

Early Detection ◽

Real Time ◽

Real Time Pcr ◽

Latent Infection ◽

Citrus Greening ◽

Pcr Assay ◽

Liberibacter Asiaticus ◽

Root Tissues ◽

Pcr Test ◽

Citrus Trees

Background: ‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus greening disease. The disease primarily involves an asymptomatic, often latent infection of CLas. However, there is no effective technique to distinguish latent-infected trees from healthy ones. This study describes the development of a new detection method for latent CLas infection using cuttings. Methods: Root tissues regenerated from cuttings using symptomatic and asymptomatic citrus trees were prepared for real-time a polymerase chain reaction (PCR) test which was used to investigate latent CLas. When some of the regenerated roots were negative for CLas in the first real-time PCR assay, a subsequent cultivation in soils was performed using the CLas-negative cuttings. CLas development during cultivation was evaluated by a second real-time PCR assay using soil-grown roots from seedlings. Results: Previously, CLas had not been detected from leaves of the latent-infected trees in our greenhouse by real-time PCR. In this study, however, CLas was detected at a moderate frequency from the root tissues of cuttings derived from the latent-infected trees, by the same PCR test. For cuttings with regenerated roots that tested negative for CLas by real-time PCR, CLas was frequently detected from roots grown in nursery soil with autoclaving, after cultivation for a month or more. Conclusions: Latent infection with CLas was detectable by real-time PCR using root tissues regenerated by cuttings and roots grown in nursery soil with autoclaving. These results suggest that the new method of investigation would provide great opportunities for early detection of CLas in asymptomatic citrus trees from field surveys, and would accelerate the eradication practice of citrus greening.

Download Full-text

A rice mTERF protein V14 sustains photosynthesis establishment and temperature acclimation in early seedling leaves

BMC Plant Biology ◽

10.1186/s12870-021-03192-2 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Man Wang ◽

Feng Zhou ◽

Hong Mei Wang ◽

De Xing Xue ◽

Yao-Guang Liu ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Posttranscriptional Regulation ◽

Expression Patterns ◽

Critical Role ◽

Temperature Acclimation ◽

Northern Blotting ◽

Protein Levels ◽

Molecular Functions

Abstract Background Plant mitochondrial transcription termination factor (mTERF) family members play important roles in development and stress tolerance through regulation of organellar gene expression. However, their molecular functions have yet to be clearly defined. Results Here an mTERF gene V14 was identified by fine mapping using a conditional albino mutant v14 that displayed albinism only in the first two true leaves, which was confirmed by transgenic complementation tests. Subcellular localization and real-time PCR analyses indicated that V14 encodes a chloroplastic protein ubiquitously expressed in leaves while spiking in the second true leaf. Chloroplastic gene expression profiling in the pale leaves of v14 through real-time PCR and Northern blotting analyses showed abnormal accumulation of the unprocessed transcripts covering the rpoB-rpoC1 and/or rpoC1-rpoC2 intercistronic regions accompanied by reduced abundance of the mature rpoC1 and rpoC2 transcripts, which encode two core subunits of the plastid-encoded plastid RNA polymerase (PEP). Subsequent immunoblotting analyses confirmed the reduced accumulation of RpoC1 and RpoC2. A light-inducible photosynthetic gene psbD was also found down-regulated at both the mRNA and protein levels. Interestingly, such stage-specific aberrant posttranscriptional regulation and psbD expression can be reversed by high temperatures (30 ~ 35 °C), although V14 expression lacks thermo-sensitivity. Meanwhile, three V14 homologous genes were found heat-inducible with similar temporal expression patterns, implicating their possible functional redundancy to V14. Conclusions These data revealed a critical role of V14 in chloroplast development, which impacts, in a stage-specific and thermo-sensitive way, the appropriate processing of rpoB-rpoC1-rpoC2 precursors and the expression of certain photosynthetic proteins. Our findings thus expand the knowledge of the molecular functions of rice mTERFs and suggest the contributions of plant mTERFs to photosynthesis establishment and temperature acclimation.

Download Full-text

Real-Time PCR Quantification of Human Cytomegalovirus DNA in Amniotic Fluid Samples from Mothers with Primary Infection

Journal of Clinical Microbiology ◽

10.1128/jcm.40.5.1767-1772.2002 ◽

2002 ◽

Vol 40 (5) ◽

pp. 1767-1772 ◽

Cited By ~ 84

Author(s):

S. Gouarin ◽

E. Gault ◽

A. Vabret ◽

D. Cointe ◽

F. Rozenberg ◽

...

Keyword(s):

Amniotic Fluid ◽

Real Time ◽

Human Cytomegalovirus ◽

Real Time Pcr ◽

Primary Infection

Download Full-text

First report on equine herpesvirus type 4 isolation in Poland – evaluation of diagnostic tools

Polish Journal of Veterinary Sciences ◽

10.2478/pjvs-2013-0069 ◽

2013 ◽

Vol 16 (3) ◽

pp. 493-500

Author(s):

G. Ploszay ◽

J. Rola ◽

M. Larska ◽

J.F. Zmudzinski

Keyword(s):

Respiratory Tract ◽

Real Time ◽

Real Time Pcr ◽

Respiratory Tract Infections ◽

Latent Infection ◽

Diagnostic Tools ◽

Upper Respiratory Tract Infections ◽

Upper Respiratory Tract ◽

Upper Respiratory ◽

Tract Infections

Abstract Upper respiratory tract infections are still a serious problem in breeding and racing horses. The most common virological factors are EHV1 and EHV4, which are both a major cause of secondary infections. High EHV4 seroprevalence in Polish horses indicates a high transmission rate of this pathogen among horses and increases the need for proper diagnostics. The aim of this study was to develop a reliable laboratory diagnostic scheme for upper respiratory tract infections and to describe the first isolation of EHV4 in Poland. Twenty one nasal swabs collected from young horses under the age of 2 years showing clinical signs of equine rhinopneumonitis were tested with duplex PCR for simultaneous detection and differentiation between EHV1/EHV4. Positive samples were then subjected to virus isolation in Vero cells. Additionally, real-time PCR was developed which allowed viral copy numbers to be quantified and enabled defining that a DNA load below 103 copies per 1 ml of the sample reflected latent infection or decline of the disease. However, the sensitivity of traditional PCR proved to be sufficient in the diagnostic of the lytic infections and allowed identification of 10 EHV4 infected horses from which 3 strains were successfully isolated in cell culture. Another four EHV4 positive results were obtained by real-time PCR; but, a high Ct (threshold cycle) and a low virus DNA copy number suggested a latent infection. This report describes the first successful isolation of EHV4 from Polish horses.

Download Full-text

Field Distribution of Wheat Stripe Rust Latent Infection Using Real-Time PCR

Plant Disease ◽

10.1094/pdis-08-11-0680 ◽

2012 ◽

Vol 96 (4) ◽

pp. 544-551 ◽

Cited By ~ 9

Author(s):

Jiahui Yan ◽

Yong Luo ◽

Tingting Chen ◽

Chong Huang ◽

Zhanhong Ma

Keyword(s):

Real Time ◽

Field Distribution ◽

Stripe Rust ◽

Real Time Pcr ◽

Latent Infection ◽

Puccinia Striiformis ◽

Gansu Province ◽

Infection Level ◽

Initial Inoculum ◽

Field Plots

Stripe rust of wheat, caused by Puccinia striiformis f. tritici, is of worldwide significance. Quantification of latent infection level is critical to estimate the potential for disease epidemics. In this study, field distribution of latent infection and the corresponding observed disease were studied in two growing seasons from 2009 to 2011 in Gangu, Gansu Province and Shangzhuang, Beijing, China. A previously developed real-time polymerase chain reaction (PCR) assay was applied to obtain the molecular disease index (MDX) to quantify the level of latent infection. At 1 to 3 weeks after leaf sampling, the observed disease indices (DX) were assessed in the corresponding experimental sites. The computer software SURFER showed that the spatial distribution patterns of MDX had a linear relationship with DX in field plots with P = 0.01. The aggregation levels of MDX correlated with those of DX in the fields. The disease foci which were correctly detected for latent infections with the real-time PCR for the Gangu and Shangzhuang field plots were 71.4 and 85.7%, respectively. The triadimefon fungicide treatment focused on the detected latent infection foci reduced both the initial inoculum and disease development, resulting in an average reduction in disease area in the field plots of 73 to 81%.

Download Full-text

Expression patterns of HMGA2 in the placenta during pregnancy

10.1101/2020.12.07.20245092 ◽

2020 ◽

Author(s):

Lars Burchardt ◽

Andrea Gottlieb ◽

Burkhard M. Helmke ◽

Werner Wosniok ◽

Wolfgang Kuepker ◽

...

Keyword(s):

Real Time ◽

Gestational Age ◽

Real Time Pcr ◽

Expression Patterns ◽

Critical Role ◽

First Trimester ◽

Postnatal Life ◽

Invasive Growth ◽

Quantitative Real Time Pcr ◽

Mesenchymal Transition

AbstractBackgroundHigh-mobility group AT-hook 2 (HMGA2) expression can be detected in many embryonic and fetal tissues but becomes down-regulated during postnatal life except for many benign and malignant tumors. In the latter case, its expression has been correlated with epithelial-mesenchymal transition and invasive growth. The placenta contributes essentially to proper development of the embryo and the fetus. In a tumor-like manner it shows rapid invasive growth during the first weeks of gestation. To address the possible role of HMGA2 during placental development, we have measured its expression throughout the prenatal period and in term placentae by mRNA quantification as well as by immunohistochemistry.MethodsExpression of HMGA2 and HPRT was measured on 89 fetal placentas, encompassing calendar gestational age of five to 41 weeks, using quantitative real time-PCR. In eleven cases in addition immunohistochemistry was used to determine the localization of HMGA2 and to compare with data obtained by quantitative real time-PCR.ResultsThe expression of HMGA2 was found to be inversely correlated with gestational age (p < 0.001). For the first part of the first trimester the level of HMGA2 is high. After that the expression shows a decline down to a baseline level where it remains until birth. HMGA2 protein was mainly detected in the nuclei of the stromal cells in the placental villi.ConclusionsDuring pregnancy, the expression of HMGA2 follows a non-linear pattern of decrease. In the first trimester, from two to three weeks after the implantation of the conceptus until the blood supply is established (hypoxic phase), the expression is high, indicating a critical role during early development and in the control of its invasive behavior, respectively.

Download Full-text