RSV Growth and Quantification by Microtitration and qRT-PCR Assays

2016 ◽  
pp. 13-32 ◽  
Author(s):  
Hayat Caidi ◽  
Jennifer L. Harcourt ◽  
Lia M. Haynes
Keyword(s):  
Qrt Pcr ◽  
Pcr Assays

scholarly journals Chorionic Villous Mesenchymal Stem Cells Derived Exosomes Deliver miR-135b-5p to Trophoblasts, Promoting their Proliferation and Invasion by Targeting TXNIP via β-Catenin Pathway

2020 ◽  
Author(s):  
Yijing Chu ◽  
Yan Zhang ◽  
Guoqiang Gao ◽  
Jun Zhou ◽  
Yang Lv ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Tissue Injury ◽  
Luciferase Reporter ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Reporter Assays ◽  
Pcr Assays ◽  
Proliferation And Invasion

Abstract Background: Human chorionic villous mesenchymal stem cells (CV-MSCs) are found to be a promising and effective treatment for tissue injury. Trophoblast dysfunction during pregnancies is significantly involved in the pathogenesis of preeclampsia (PE). This work was to understand how CV-MSCs regulated trophoblast function. Methods: In this study, we treated trophoblasts with CV-MSC-derived exosomes and RNA-seq analysis was used to understand the changes in trophoblasts. We examined the levels of TXNIP and β-catenin in trophoblasts by immunohistochemistry, western blot and qRT-PCR assays. Luciferase reporter assays and qRT-PCR assays were used to understand the role of miR135b-5p in the effects of CV-MSC-derived exosomes. The growth and invasion of trophoblasts was evaluated with the CCK-8 and transwell assays. Results: The treatment markedly enhanced the trophoblast proliferation and invasion. Furthermore, a significant decrease of TXNIP expression and inactivation of the β-catenin pathway in CV-MSCs exosomes-treated trophoblasts was observed. Consistent with these findings, TXNIP inhibition exhibited the same effect of promoting trophoblast proliferation and invasion as induced by CV-MSC-derived exosomes, also with the accompaniment of inactivation of β-catenin pathway. In addition, overexpression of TXNIP activated the β-catenin pathway in trophoblasts, and reduced the proliferation and invasion of trophoblasts. Importantly, miR135b-5p was found to be highly expressed in CV-MSC exosomes and interact with TXNIP. The miR-135b-5p overexpression significantly elevated the proliferation and invasion of trophoblasts, which could be attenuated by TXNIP overexpression. Conclusion: Our results suggest that TXNIP-dependent β-catenin pathway inactivation mediated by miR135b-5p which is delivered by CV-MSC-derived exosomes could promote the proliferation and invasion of trophoblasts.

scholarly journals A New Hendra Virus Genotype Found in Australian Flying Foxes

2021 ◽  
Author(s):  
Jianning Wang ◽  
Danielle E Anderson ◽  
Kim Halpin ◽  
Xiao Hong ◽  
Honglei Chen ◽  
...  
Keyword(s):  
Genetic Relatedness ◽  
Phylogenetic Analyses ◽  
Inflammatory Lesion ◽  
Hendra Virus ◽  
Virus Genotype ◽  
Flying Foxes ◽  
Qrt Pcr ◽  
Genotype 2 ◽  
Pcr Assays ◽  
Flying Fox

Abstract Background Hendra virus (HeV) has caused lethal disease outbreaks in humans and horses in Australia. Pteropid bats (flying foxes) are the wildlife reservoir from which the virus was first isolated in 1996. Following a heat stress mortality event in Australian flying foxes in 2013, a novel HeV variant was discovered. This study describes the subsequent surveillance of Australian flying foxes for this novel virus over a nine year period using qRT-PCR testing of bat tissues submitted primarily for Australian bat lyssavirus (ABLV) diagnosis. Genome sequencing and characterisation of the novel HeV variant was also undertaken. Methods Spleen and kidney samples harvested from flying fox carcasses were initially screened with two real-time qRT-PCR assays specific for the prototype HeV. Two additional qRT-PCR assays were developed specific for the HeV variant first detected in samples from a flying fox in 2013. Next-generation sequencing and virus isolation was attempted from selected samples to further characterise the new virus. Results Since 2013, 98 flying foxes were tested and 11 were positive for the new HeV variant. No samples were positive for the original HeV. Ten of the positive samples were from grey-headed flying foxes (GHFF, Pteropus poliocephalus), however this species was over-represented in the opportunistic sampling (83% of bats tested were GHFF). The positive GHFF samples were collected from Victoria and South Australia and one positive Little red flying fox (LRFF, Pteropus scapulatus) was collected from Western Australia. Immunohistochemistry (IHC) confirmed the presence of henipavirus antigen, associated with an inflammatory lesion in cardiac blood vessels of one GHFF. Positive samples were sequenced and the complete genome was obtained from three samples. When compared to published HeV genomes, there was 84% sequence identity at the nucleotide level. Based on phylogenetic analyses, the newly detected HeV belongs to the HeV species but occupies a distinct lineage. We have therefore designated this virus HeV genotype 2 (HeV-G2). Attempts to isolate virus from PCR positive samples have not been successful. Conclusions A novel HeV genotype (HeV-G2) has been identified in two flying fox species submitted from three states in Australia, indicating that the level of genetic diversity for HeV is broader than first recognised. Given its high genetic relatedness to HeV, HeV-G2 should be considered a zoonotic pathogen.

scholarly journals Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis

2005 ◽  
Vol 109 (4) ◽  
pp. 365-379 ◽  
Author(s):  
Stephen A. Bustin ◽  
Reinhold Mueller
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Therapy Monitoring ◽  
Clinical Diagnostics ◽  
Clinical Diagnostic Laboratory ◽  
Diagnostic Laboratory ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Clinical Diagnostic ◽  
Pcr Assays

qRT-PCR (real-time reverse transcription-PCR) has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in novel clinical diagnostic assays. Quantitative results obtained by this technology are not only more informative than qualitative data, but simplify assay standardization and quality management. qRT-PCR assays are most established for the detection of viral load and therapy monitoring, and the development of SARS (severe acute respiratory syndrome)-associated coronavirus qRT-PCR assays provide a textbook example of the value of this technology for clinical diagnostics. The widespread use of qRT-PCR assays for diagnosis and the detection of disease-specific prognostic markers in leukaemia patients provide further examples of their usefulness. Their value for the detection of disease-associated mRNA expressed by circulating tumour cells in patients with solid malignancies is far less apparent, and the clinical significance of results obtained from such tests remains unclear. This is because of conceptual reservations as well as technical limitations that can interfere with the diagnostic specificity of qRT-PCR assays. Therefore, although it is evident that qRT-PCR assay has become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations if it is to fulfil its potential.

scholarly journals Selection of internal references for qRT-PCR assays of human hepatocellular carcinoma cell lines

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Yang Liu ◽  
Zhaoyu Qin ◽  
Lili Cai ◽  
Lili Zou ◽  
Jing Zhao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Reference Genes ◽  
Systematic Evaluation ◽  
Sorafenib Treatment ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Carcinoma Cell Lines ◽  
Pcr Assays

Selecting internal references is important for normalizing the loading quantity of samples in quantitative reverse-transcription PCR (qRT-PCR). In the present study, a systematic evaluation of reference genes among nine hepatocellular carcinoma (HCC) cell lines was conducted. After screening the microarray assay data of ten HCC cell lines, 19 candidate reference genes were preselected and then evaluated by qRT-PCR, together with ACTB, GAPDH, HPRT1 and TUBB. The expression evenness of these candidate genes was evaluated using RefFinder. The stabilities of the reference genes were further evaluated under different experimental perturbations in Huh-7 and MHCC-97L, and the applicability of the reference genes was assessed by measuring the mRNA expression of CCND1, CCND3, CDK4 and CDK6 under sorafenib treatment in Huh-7. Results showed that TFG and SFRS4 are among the most reliable reference genes, and ACTB ranks third and acts quite well as a classical choice, whereas GAPDH, HPRT1 and TUBB are not proper reference genes in qRT-PCR assays among the HCC cell lines. SFRS4, YWHAB, SFRS4 and CNPY3 are the most stable reference genes of the MHCC-97L under the perturbations of chemotherapy, oxidative stress, starvation and hypoxia respectively, whereas YWHAB is the most stable one of Huh-7 under all perturbations. GAPDH is recommended as a reference gene under chemotherapy perturbations. YWHAB and UBE2B, TMED2 and TSFM, and GAPDH and TSFM are the two best reference genes under oxidative stress, starvation and hypoxia perturbations respectively. TSFM is stable in both cell lines across all the perturbations.

scholarly journals Development and Validation of Heminested RT-PCR and qRT-PCR Assays for Comprehensive Detection of Rabies Virus in the Suspected Rabid Brain and Saliva Samples

2019 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Fereshteh Amiri ◽  
Zohreh Fadajan ◽  
Azadeh Rasooli ◽  
Iman Salahshourifar ◽  
Rouzbeh Bashar ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Rt Pcr ◽  
Qrt Pcr ◽  
Pcr Assays ◽  
Development And Validation

scholarly journals Comparison of three TaqMan Real-Time Reverse Transcription-PCR assays in detecting SARS-CoV-2

2020 ◽  
Author(s):  
Yan Xiao ◽  
Zhen Li ◽  
Xinming Wang ◽  
Yingying Wang ◽  
Ying Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Detection Rate ◽  
N Gene ◽  
Detection Rates ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Linear Detection ◽  
Gene Assay ◽  
Pcr Assays

AbstractQuick and accurate detection of SARS-CoV-2 is critical for COVID-19 control. Dozens of real-time reverse transcription PCR (qRT-PCR) assays have been developed to meet the urgent need of COVID-19 control. However, methodological comparisons among the developed qRT-PCR assays are limited. In the present study, we evaluated the sensitivity, specificity, amplification efficiency, and linear detection ranges of three qRT-PCR assays, including the assays developed by our group (IPBCAMS), and the assays recommended by WHO and China CDC (CCDC). The three qRT-PCR assays exhibited similar sensitivities, with the limit of detection (LOD) at about 10 copies per reaction (except the ORF 1b gene assay in CCDC assays with a LOD at about 100 copies per reaction). No cross reaction with other respiratory viruses were observed in all of the three qRT-PCR assays. Wide linear detection ranges from 106 to 101 copies per reaction and acceptable reproducibility were obtained. By using 25 clinical specimens, the N gene assay of IPBCAMS assays and CCDC assays performed better (with detection rates of 92% and 100%, respectively) than that of the WHO assays (with a detection rate of 60%), and the ORF 1b gene assay in IPBCAMS assays performed better (with a detection rate of 64%) than those of the WHO assays and the CCDC assays (with detection rates of 48% and 20%, respectively). In conclusion, the N gene assays of CCDC assays and IPBCAMS assays and the ORF 1b gene assay of IPBCAMS assays were recommended for qRT-PCR screening of SARS-CoV-2.

scholarly journals Evaluation of WHO listed COVID-19 qPCR primers and probe in silico with 375 SERS-CoV-2 full genome sequences

2020 ◽  
Author(s):  
Derek Toms ◽  
Julang Li ◽  
Hugh Y. Cai
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
World Health ◽  
Sequence Information ◽  
Genome Sequences ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Successful Design ◽  
Pcr Assays ◽  
Health Organization

AbstractQuantitative reverse-transcription PCR (qRT-PCR) assays remains the gold standard for detection of the SARS-CoV-2 virus because of its sensitivity and specificity. However, successful design of qRT-PCR assays requires accurate viral genome sequences. With mutations accumulating as the virus is transmitted globally, we sought to compare current assays recommended by the World Health Organization with available SARS-CoV-2 genomic sequences in silico. While most sequences were conserved, there were notable mismatches, particularly in assays developed using early sequences when compared to more recent isolates. We recommend that any assay being evaluated for diagnostic tests be compared with prevalent sequence data from the region of proposed testing and that continued publicly accessible sequence information continue to be provided by the research community.

Differential Expression Profiles of Long Noncoding RNA and mRNA of Osteogenically Differentiated Mesenchymal Stem Cells in Ankylosing Spondylitis

2016 ◽  
Vol 43 (8) ◽  
pp. 1523-1531 ◽  
Author(s):  
Zhongyu Xie ◽  
Jinteng Li ◽  
Peng Wang ◽  
Yuxi Li ◽  
Xiaohua Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ankylosing Spondylitis ◽  
Osteogenic Differentiation ◽  
Noncoding Rna ◽  
Bioinformatics Analysis ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Qrt Pcr ◽  
Pcr Assays

Objective.We previously demonstrated that mesenchymal stem cells (MSC) from patients with ankylosing spondylitis (AS; ASMSC) have a greater osteogenic differentiation capacity than MSC from healthy donors (HDMSC) and that this difference underlies the pathogenesis of pathological osteogenesis in AS. Here we compared expression levels of long noncoding RNA (lncRNA) and mRNA between osteogenically differentiated ASMSC and HDMSC and explored the precise mechanism underlying abnormal osteogenic differentiation in ASMSC.Methods.HDMSC and ASMSC were induced with osteogenic differentiation medium for 10 days. Microarray analyses were then performed to identify lncRNA and mRNA differentially expressed between HDMSC and ASMSC, which were then subjected to bioinformatics analysis and confirmed by quantitative real-time PCR (qRT-PCR) assays. In addition, coding-non-coding gene co-expression (CNC) networks were constructed to examine the relationships between the lncRNA and mRNA expression patterns.Results.A total of 520 lncRNA and 665 mRNA were differentially expressed in osteogenically differentiated ASMSC compared with HDMSC. Bioinformatics analysis revealed 64 signaling pathways with significant differences, including transforming growth factor-β signaling. qRT-PCR assays confirmed the reliability of the microarray data. The CNC network indicated that 4 differentially expressed lncRNA, including lnc-ZNF354A-1, lnc-LIN54-1, lnc-FRG2C-3, and lnc-USP50-2 may be involved in the abnormal osteogenic differentiation of ASMSC.Conclusion.Our study characterized the differential lncRNA and mRNA expression profiles of osteogenically differentiated ASMSC and identified 4 lncRNA that may participate in the abnormal osteogenic differentiation of ASMSC. These results provide insight into the pathogenesis of pathological osteogenesis in AS.

scholarly journals Construction of prcK and prcR Mutant Strains of Lactobacillus paracasei HD1.7 and the Impact on the Production of Paracin 1.7

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Jingping Ge ◽  
Xiaolei Ji ◽  
Tian You ◽  
Yanyang Sun ◽  
Wenxiang Ping
Keyword(s):  
Quorum Sensing ◽  
Antimicrobial Peptides ◽  
Parental Strain ◽  
Antimicrobial Activities ◽  
Qrt Pcr ◽  
Mutant Strains ◽  
Gene Knockouts ◽  
Pcr Assays ◽  
Chemical Preservatives ◽  
The Impact

Gene knockouts of prcK, prcR and both together were constructed in L. paracasei HD1.7. The antimicrobial activities of the prcK, prcR and prcKprcR mutant strains against B. subtilis were 23.6%, 21.9% and 36.6% lower than that of the parental strain, respectively, indicating that these genes affect production of bacteriocin antimicrobial peptides. qRT-PCR assays showed that the relative transcription levels of prcK and prcR mRNA in the DK and DR strains were 0.36 and 0.33 times of that in parental bacteria, respectively. Our data suggest that prcK and prcR are quorum sensing related genes that influence production of the bacteriocin Paracin 1.7. This research provides the basis for exploring the functions of these genes in the production of Paracin 1.7 and more generally for the exploration of the biological preservatives instead of chemical preservatives.

scholarly journals DNA and RNA References for qRT-PCR Assays in Exfoliated Cervical Cells

2006 ◽  
Vol 8 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Martin Steinau ◽  
Mangalathu S. Rajeevan ◽  
Elizabeth R. Unger
Keyword(s):  
Qrt Pcr ◽  
Dna And Rna ◽  
Pcr Assays ◽  
Cervical Cells
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