Usefulness of a real-time PCR platform for G+C content and DNA–DNA hybridization estimations in vibrios

The aim of this study was to evaluate the utility of a real-time PCR platform to estimate the DNA G+C content (mol%) and DNA–DNA hybridization (DDH) values in the genus Vibrio. In total, nine vibrio strains were used to determine the relationship between genomic DNA G+C content and T m (°C). The T m and HPLC datasets fit a linear regression curve with a significant correlation coefficient, corroborating that this methodology has a high correlation with the standard methodology based on HPLC (R2 = 0.94). Analysis of 31 pairs of vibrios provided a wide range of ΔT m values, varying between 0.72 and 12.5 °C. Pairs corresponding to strains of the same species or strains from sister species showed the lowest ΔT m values. For instance, the ΔT m of the sister species Vibrio harveyi LMG 4044T and Vibrio campbellii LMG 11216T was 5.2 °C, whereas the ΔT m of Vibrio coralliilyticus LMG 20984T and Vibrio neptunius LMG 20536T was 8.75 °C. The mean ΔT m values corresponding to pairs of strains with DDH values lower than 60 % or higher than 80 % were, respectively, 8.29 and 2.21 °C (significant difference, P<0.01). The high correlation between DDH values obtained in previous studies and the ΔT m values (R2 = 0.7344) indicates that the fluorimetric methodology is a reliable alternative for the estimation of both DNA G+C content and ΔT m in vibrios. We suggest that strains of the same Vibrio species will have less than 4 °C ΔT m. The use of a real-time PCR platform represents a valuable alternative for the development of the taxonomy of vibrios.

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Real-Time PCR Gene Expression Profiling of Microarray Gene Signatures in Acute Leukemia.

Blood ◽

10.1182/blood.v104.11.4401.4401 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4401-4401

Author(s):

Ebrahim Sakhinia ◽

Mahboubeh Farahangpour ◽

John A. Liu Yin ◽

Gerard Brady ◽

Judith A. Hoyland ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Cyclin D3 ◽

Expression Level ◽

Tumor Type ◽

Gene Signatures ◽

Significant Difference ◽

The Mean ◽

Microarray Gene

Abstract Cancer subtype discovery and classification using microarray gene signatures has the potential to transform pathological diagnosis but measurement of indicator genes in routine practice remains difficult. We tested use of real-time PCR measurement of indicator genes for AML and ALL (Golub et al, Science, 1999) as a method for validation and application of microarray gene signatures. Mononuclear cells (MC) were isolated from whole bone marrow (BM) aspirates by density gradient centrifugation and sorted into unselected (total), CD34+ve and CD34-ve fractions. The mRNA in each fraction was globally amplified using a PolyA PCR method. We measured the expression profile of the 17 top ranked genes (cystatin C, leptin receptor, fumarylacetoacetate, CD33, HoxA9, adipsin, proteoglycan 1, LTC4 synthase, LYN, C-myb, MB-1, cyclin D3, SNF2, RbAp48, proteasome iota, HkrT-1 and E2A) from Golub et al (1999) by real-time PCR. All values were calibrated against control standards and normalized to the mean of three housekeeping genes (IF2-beta, GAPDH and human ribosomal protein S9). Data for all 17 genes were obtained for 4 (ALL), 26 (AML), 12 (AML remission) and 9 (morphologically normal) BM samples, each fractionated into three fractions (total MC, CD34+ve MC & CD34−ve MC). There was no significant difference in the mean of three housekeeping gene expression levels between the diagnostic groups. Comparison of the expression level of the other genes confirmed ability to separate AML and ALL, whilst the direction of expression change (increased or decreased) for each gene between AML and ALL was the same as found by Golub et al. In particular, c-myb showed largest significant increase in ALL vs AML in the total BM fraction, whilst cystain c was increased in AML in the CD34−ve fraction. hSNF2b was significantly increased in the ALL total B.M fraction and Hox-A9 was significantly increased in the AML CD34+ve B.M fraction. Furthermore expression level of LYN and CD33 was significantly increased in AML compared to remission AML, indicating ability of the method to determine activity status of disease. In addition, several of the genes provided better separation between AML and ALL when measured in the CD34+ve and −ve fractions indicating more prominent expression in cells of different maturity and that prior fractionation is diagnostically more informative. The results demonstrate ability of the method to validate gene expression signatures by an independent method, which is simple, sensitive and robust, allowing translation to routine clinical use. Whilst the present study used AML and ALL, in principle the method could be extended to any other tumor type for which gene signatures exist.

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Evaluation of Three Real-Time PCR Methods for Detection of Salmonella from Cloves

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-498 ◽

2017 ◽

Vol 80 (6) ◽

pp. 982-989 ◽

Cited By ~ 7

Author(s):

Aparna Tatavarthy ◽

Laila Ali ◽

Vikas Gill ◽

Lijun Hu ◽

Xiaohong Deng ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Extraction Methods ◽

Pcr Assay ◽

Wide Range ◽

The Mean ◽

Salmonella Serotypes ◽

Pathogen Dna ◽

Dna Extraction Methods

ABSTRACTThe purpose of the study was to evaluate three real-time PCR platforms for rapid detection of Salmonella from cloves and to compare three different DNA extraction methods. Six trials were conducted with two clove cultivars, Ceylon and Madagascar, and three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Each trial consisted of 20 test portions. The preenrichment cultures were used to perform PCR for comparison of the effectiveness of U.S. Food and Drug Administration, Pacific Regional Laboratory Southwest (FDA-PRLSW), Applied Biosystems Inc. (ABI) MicroSEQ, and GeneDisc platforms for detection of Salmonella. Three DNA extraction methods were used: standard extraction method for each PCR platform, boil preparation, and LyseNow food pathogen DNA extraction cards. The results from real-time PCR correlated well with FDA Bacteriological Analytical Manual culture assay results, with a wide range of cycle threshold (CT) values among the three PCR platforms for intended positive samples. The mean CT values for MicroSEQ (16.36 ± 2.78) were significantly lower than for PRLSW (20.37 ± 3.45) and GeneDisc (23.88 ± 2.90) (P < 0.0001). Pairwise comparisons between PCR platforms using different DNA extraction methods indicate that the CT values are inversely proportional to the relative DNA quantity (RDQ) yields by different platform-extraction combinations. The pairing of MicroSEQ and boil preparation generated the highest RDQ of 120 and the lowest average CT value of 14.48, whereas the pairing of GeneDisc and LyseNow generated the lowest RDQ of 0.18 and the highest average CT of 25.97. Boil preparation yielded higher RDQ than the other extraction methods for all three PCR platforms. Although the MicroSEQ platform generated the lowest CT values, its sensitivity was compromised by narrow separations between the positive and negative samples. The PRLSW platform generated the best segregation between positive and negative groups and is less likely to produce false results. In conclusion, FDA-PRLSW was the most efficient PCR assay for Salmonella detection, and boil preparation was the best method for DNA extraction.

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Saliva Quantification of SARS-CoV-2 in Real-Time PCR From Asymptomatic or Mild COVID-19 Adults

Frontiers in Microbiology ◽

10.3389/fmicb.2021.786042 ◽

2022 ◽

Vol 12 ◽

Author(s):

Florence Carrouel ◽

Emilie Gadea ◽

Aurélie Esparcieux ◽

Jérome Dimet ◽

Marie Elodie Langlois ◽

...

Keyword(s):

Viral Load ◽

Real Time ◽

Real Time Pcr ◽

Clinical Symptoms ◽

Age Groups ◽

Nasal Discharge ◽

Viral Loads ◽

Co Morbidity ◽

Significant Difference ◽

The Relationship

The fast spread of COVID-19 is related to the highly infectious nature of SARS-CoV-2. The disease is suggested to be transmitted through saliva droplets and nasal discharge. The saliva quantification of SARS-CoV-2 in real-time PCR from asymptomatic or mild COVID-19 adults has not been fully documented. This study analyzed the relationship between salivary viral load on demographics and clinical characteristics including symptoms, co-morbidities in 160 adults diagnosed as COVID-19 positive patients recruited between September and December 2020 in four French centers. Median initial viral load was 4.12 log10 copies/mL (IQR 2.95–5.16; range 0–10.19 log10 copies/mL). 68.6% of adults had no viral load detected. A median load reduction of 23% was observed between 0–2 days and 3–5 days, and of 11% between 3–5 days and 6–9 days for the delay from onset of symptoms to saliva sampling. No significant median difference between no-symptoms vs. symptoms patients was observed. Charge was consistently similar for the majority of the clinical symptoms excepted for headache with a median load value of 3.78 log10 copies/mL [1.95–4.58] (P < 0.003). SARS-CoV-2 RNA viral load was associated with headache and gastro-intestinal symptoms. The study found no statistically significant difference in viral loads between age groups, sex, or presence de co-morbidity. Our data suggest that oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.

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Intracranial Pressure during External Ventricular Drainage Weaning Is an Outcome Predictor of Traumatic Brain Injury

BioMed Research International ◽

10.1155/2020/8379134 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Jia-cheng Gu ◽

Hong Wu ◽

Xing-zhao Chen ◽

Jun-feng Feng ◽

Guo-yi Gao ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Brain Injury ◽

Intracranial Pressure ◽

External Ventricular Drainage ◽

Unfavorable Outcome ◽

Ventricular Drainage ◽

Outcome Group ◽

Significant Difference ◽

The Mean ◽

The Relationship

External ventricular drainage (EVD) is widely used in patients with a traumatic brain injury (TBI). However, the EVD weaning trial protocol varies and insufficient studies focus on the intracranial pressure (ICP) during the weaning trial. We aimed to establish the relationship between ICP during an EVD weaning trial and the outcomes of TBI. We enrolled 37 patients with a TBI with an EVD from July 2018 to September 2019. Among them, 26 were allocated to the favorable outcome group and 11 to the unfavorable outcome group (death, post-traumatic hydrocephalus, persistent vegetative state, and severe disability). Groups were well matched for sex, pupil reactivity, admission Glasgow Coma Scale score, Marshall computed tomography score, modified Fisher score, intraventricular hemorrhage, EVD days, cerebrospinal fluid output before the weaning trial, and the complications. Before and during the weaning trial, we recorded the ICP at 1-hour intervals to calculate the mean ICP, delta ICP, and ICP burden, which was defined as the area under the ICP curve. There were significant between-group differences in the age, surgery types, and intensive care unit days (p=0.045, p=0.028, and p=0.004, respectively). During the weaning trial, 28 (75.7%) patients had an increased ICP. Although there was no significant difference in the mean ICP before and during the weaning trial, the delta ICP was higher in the unfavorable outcome group (p=0.001). Moreover, patients who experienced death and hydrocephalus had a higher ICP burden, which was above 20 mmHg (p=0.016). Receiver operating characteristic analyses demonstrated the predictive ability of these variables (area under the curve AUC=0.818 [p=0.002] for delta ICP and AUC=0.758 [p=0.038] for ICP burden>20 mmHg). ICP elevation is common during EVD weaning trials in patients with TBI. ICP-related parameters, including delta ICP and ICP burden, are significant outcome predictors. There is a need for larger prospective studies to further explore the relationship between ICP during EVD weaning trials and TBI outcomes.

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Effects of Language Learning Strategies and Learners’ Motivation on Students’ Learning Achievement

International Journal of English Language Education ◽

10.5296/ijele.v3i2.8276 ◽

2015 ◽

Vol 3 (2) ◽

pp. 132 ◽

Cited By ~ 1

Author(s):

Hao-Yuan Cheng ◽

Nai-Ying Chang

Keyword(s):

Language Learning ◽

Learning Strategies ◽

Language Learning Strategies ◽

Course Performance ◽

Test Analysis ◽

New Information ◽

Significant Difference ◽

Group A ◽

The Mean ◽

The Relationship

<p>This study investigated whether learning strategies had made any impact on learners’ achievement and explored whether learning motivation was correlated with learning strategies. The participants of this study were the students from the EFL (English as a Foreign Language) intermediate level course at a college in Taiwan. The students were given a pretest and a posttest. The mean scores of these tests were compared with a SILL survey (Strategies Inventory for Language Learning) at the end of the investigation. The participants’ course performance was compared with their use of learning strategies. The assumption of the relationship between learning strategies and motivation is that motivated learners have a greater desire to seek out solutions or support from others and employ more strategies to process the new information. The results of this investigation revealed that only the memory strategies had a significant difference in the posttest of Group A on the independent sample t-test analysis.</p>

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Genome-wide screening of m6A-modified transcript in the Wilms’ tumor

10.21203/rs.3.rs-197376/v1 ◽

2021 ◽

Author(s):

Zhuo Liu ◽

Feng He ◽

Jing Liu ◽

Shengrong OuYang ◽

Zexi Li ◽

...

Keyword(s):

Gene Ontology ◽

Real Time ◽

Real Time Pcr ◽

Wilms Tumor ◽

Expression Patterns ◽

Mrna Levels ◽

Gene Ontology Analysis ◽

Quantitative Real Time Pcr ◽

Related Factors ◽

Significant Difference

Abstract Background Wilms’ tumor, also called nephroblastoma, is the most common pediatric renal malignancy. The pathogenesis of Wilms’ tumor has been attributed to several genetic and epigenetic factors. However, the most pervasive internal mRNA modification that affects almost every process of RNA metabolism, RNA N6-Methyladenosine (m6A) methylation, has not been characterized in Wilms’ tumor. Methods Wilms’ tumor (WT) and adjacent non-cancerous (NC) tissue samples were obtained from 23 children with nephroblastoma, and the global m6A levels were measured by mass spectrometry. Analyses by m6A-mRNA epitranscriptomic microarray and mRNA microarray were performed, and m6A-related mRNAs were validated by quantitative real-time PCR for input and m6A-immunoprecipitated RNA samples from WT and NC tissues. Gene ontology analysis and KEGG pathway analysis were performed for differentially expressed genes, and expression of RNA methylation-related factors was measured by quantitative real-time PCR. Results The total m6A methylation levels in total RNA of WT samples and NC samples were (0.21 ± 0.01)% and (0.22 ± 0.01)%, respectively, with no statistically significant difference. Fifty-nine transcripts were differentially m6A-methylated between the WT and NC groups, which showed distinct m6A modification patterns. Gene ontology analysis indicated that m6A-modified genes were enriched in cancer-associated pathways, including the mTOR pathway, and conjoint analysis of the unique methylation and gene expression patterns in WT samples suggested an association with metabolic pathways.The mRNA levels of the m6A-related “reader” genes, YTHDF1, YTHDF2 and IGF2BP3, were statistically higher in WT samples than in NC samples. Conclusion This is the first study to determine the m6A modification profiles in Wilms’ tumor. Our data provide novel information regarding patterns of m6A modification that correlate with carcinogenesis in Wilms’ tumor.

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The relationship between workload and adherence to professional codes of ethics among a sample of Iranian nurses

Clinical Ethics ◽

10.1177/14777509211040183 ◽

2021 ◽

pp. 147775092110401

Author(s):

Mahsa Dadkhah-Tehrani ◽

Mohsen Adib-Hajbaghery

Keyword(s):

Personal Characteristics ◽

Codes Of Ethics ◽

Spearman Correlation ◽

Cross Sectional ◽

Significant Difference ◽

Perceived Workload ◽

Professional Codes ◽

The Mean ◽

Professional Codes Of Ethics ◽

The Relationship

Background Many studies have investigated the adherence to professional codes of ethics by nurses. However, no study has explicitly examined the relationship between workload and adherence to professional codes of ethics among Iranian nurses. Objective This study aimed to explore the relationships between workload and adherence to professional codes of ethics among a sample of Iranian nurses. Materials and Methods A cross-sectional descriptive study was conducted on 213 nurses who were randomly selected from the different wards of Shahid Beheshti Hospital of Kashan, Iran. Data collection instruments included a personal characteristics questionnaire, the Nursing Ethics Questionnaire, and the National Aeronautics and Space Administration Task Load Index. The Chi-square, the Spearman correlation coefficient, the Kruskal–Wallis, and Mann–Whitney U tests were used to analyze the data. Results A majority of nurses were females (77.5%), married (79.3%), and permanently employed (55.4%). The mean of the overall perceived workload and adherence to professional codes of ethics were 76.36 ± 13.64 and 13.98 ± 2.58, that were at high and moderated level, respectively. Spearman correlation test showed no significant correlation between perceived workload and adherence to professional codes of ethics ( r = 0.03, P = 0.6). A significant difference was found between the mean scores of adherence to professional codes of ethics in nurses working in different departments ( P < 0.001). Conclusion Participating nurses perceived high levels of workload but their mean adherence to professional codes of ethics was at a moderate level. The perceived workload was indirectly correlated with adherence to professional codes of ethics.

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Building a molecular typing protocol for rs1801133 based on real-time PCR HRM technique

Science and Technology Development Journal - Natural Sciences ◽

10.32508/stdjns.v2i3.747 ◽

2019 ◽

Vol 2 (3) ◽

pp. 5-13

Author(s):

Linh Thi Nhut Tran ◽

My Thi Huynh Nguyen ◽

Linh Nguy Hoang Le ◽

Khoa Dang Le ◽

Minh Hoang Nhat Nguyen ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Neurological Diseases ◽

Human Diseases ◽

Chromosome 1 ◽

Nucleotide Sequencing ◽

Sequencing Method ◽

Mgcl2 Concentration ◽

Clear Differentiation ◽

The Relationship

rs1801133 is a single nucleotide polymorphism (SNP) located in the sequence of MTHFR on human chromosome 1. The alleles of this SNP affect the activity of the MTHFR enzyme. People bearing C/T genotype have 66% activity of MTHFR while people with T/T genotype have only 25% activity. These reduced activities of MTHFR cause homocysteinemia. There are several publications on the relationship between homocysteinemia and human diseases such as cardiovascular disease, neurological diseases, abnormal fetus, infertility and cancer. In this study, we built a molecular protocol for genotyping rs1801133 using real-time PCR HRM technique. This protocol could be used for diagnosis of molecular mechanism of homocysteinemia causing the mentoned above diseases as well as for the study of the relationship between rs1801133 and other human diseases. We successfully designed the primer pairs for genotyping and nucleotide sequencing rs1801133 by real-time PCR HRM and Sanger sequencing method. We also examined the optimal MgCl2 concentration for clear differentiation of three rs1801133 genotypes. Performance characteristics of the real-time PCR HRM protocol included of specificity, repeatability, reproducibility was evaluated and it showed good results. Comparison of genotyping results of rs1801133 between the realtime PCR HRM method and the Sanger nucleotide sequencing method showed good concordances. Finally, this real-time PCR HRM protocol for rs1801133 genotyping was applied on 100 human DNA samples to evaluate the clinical utility of the protocol.

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The association of male infertility with telomere length: A case control study

Indian Journal of Clinical Anatomy and Physiology ◽

10.18231/j.ijcap.2021.070 ◽

2021 ◽

Vol 8 (4) ◽

pp. 325-332

Author(s):

Kate Deepali Rajesh ◽

Puranam Vatsalaswamy ◽

Manvikar Purshotam Rao

Keyword(s):

Male Infertility ◽

Telomere Length ◽

Real Time ◽

Real Time Pcr ◽

Case Control Study ◽

Case Control ◽

Control Group ◽

Significant Difference ◽

Control Study ◽

Sperm Telomere Length

To study the relevance of sperm telomere length and infertility in men. : Our case-control study included twenty-five males in couple with sub-fertility/infertility (test group) and twenty five healthy males (control group) with proven paternity in the age group 25 to 35 years. The Absolute Sperm Telomere length (aSTL) was measured by real-time PCR. We investigated whether any significant difference in the aSTL value existed between the groups and analysed the relationship between aSTL and other sperm parameters.The mean (SE) aSTL recorded in the infertile cases was significantly shorter than for the control group being 140.60 (6.66) Kb/genome and 239.63 (12.32) Kb/genome respectively (p <0.001) A weak correlation was eminent between aSTL kb/genome and the total sperm count mil/ml (rho= 0.04, p - 0.86), progressive sperm motility (rho= - 0.02, p=0.934) and sperm viability (rho= - 0.07 p=0.741) in the infertile group. The measurement of aSTL by real-time PCR is a simple and rapid method that offers further paramount information with respective to the quality of sperm. It is befitted for epidemiological studies, hence opening new perspectives in the evaluation of male infertility. Limitations - Our study was confined to men aged between 25 and 35 years. Further comparative studies are needed to explore the significance of STL and infertility in older males. Additional studies will help illumine the significance of aSTL as a prognostic biomarker in assisted reproduction.

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Relationship between Growth and Wood properties in Agathis sp. Planted in Indonesia

Wood Research Journal ◽

10.51850/wrj.2012.3.1.1-5 ◽

2017 ◽

Vol 3 (1) ◽

pp. 1-5

Author(s):

Futoshi Ishiguri ◽

Kazuko Makino ◽

Imam Wahyudi ◽

Jun Tanabe ◽

Yuya Takashima ◽

...

Keyword(s):

Wood Quality ◽

Basic Density ◽

Tree Breeding ◽

Wood Properties ◽

Stem Diameter ◽

Breeding Programs ◽

Significant Difference ◽

Pilodyn Penetration ◽

The Mean ◽

The Relationship

The present study clarified the relationship between the growth and wood properties of 54-year-old Agathis sp. trees planted in Indonesia. Stem diameter, pilodyn penetration, and stress-wave velocity (SWV) were measured for all trees (35 trees) in a plot (30  30 m) located almost at the center of a stand. Based on the mean stem diameter, 10 standard trees in a plot were selected for measuring the basic density (BD) and compressive strength parallel to grain (CS). Core samples (5 mm in diameter) were collected from the 10 selected trees to determine BD and CS. The mean stem diameter, pilodyn penetration and SWV in the plot were 40.2  11.3 cm, 23.4  2.1 cm, and 3.85  0.43 km/s, respectively. No significant correlation coefficeint (r = -0.327, no significance at 5% level) was obtained between stem diameter and SWV. The mean BD and CS in the 10 trees were 0.42  0.03 g/cm3 and 28.1  2.7 MPa, respectively. A significant positive correlation was observed between BD and CS. Analysis of variance (ANOVA) revealed a significant difference between BD and CS values of the 10 trees, indicating that wood properties may differ among trees with the same standard growth in a stand. From these results, we concluded that wood quality improvement in this species could be achieved by selecting trees with high density and strength in tree breeding programs.

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