scholarly journals A set of RT-PCR assays for detection of all known avian paramyxoviruses and application in surveillance of avian paramyxoviruses in China

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10748
Author(s):  
Ji-Hui Jin ◽  
Jing-Jing Wang ◽  
Ying-Chao Ren ◽  
Shuo Liu ◽  
Jin-Ping Li ◽  
...  
Keyword(s):  
Large Scale ◽  
Class Ii ◽  
Class I ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Degenerate Primers ◽  
Double Stranded Rna ◽  
Live Poultry ◽  
Pcr Assays

Background Avian paramyxoviruses (APMVs), also termed avian avulaviruses, are of a vast diversity and great significance in poultry. Detection of all known APMVs is challenging, and distribution of APMVs have not been well investigated. Methods A set of reverse transcription polymerase chain reaction (RT-PCR) assays for detection of all known APMVs were established using degenerate primers targeting the viral polymerase L gene. The assays were preliminarily evaluated using in-vitro transcribed double-stranded RNA controls and 24 known viruses, and then they were employed to detect 4,346 avian samples collected from 11 provinces. Results The assays could detect 20–200 copies of the double-stranded RNA controls, and detected correctly the 24 known viruses. Of the 4,346 avian samples detected using the assays, 72 samples were found positive. Of the 72 positives, 70 were confirmed through sequencing, indicating the assays were specific for APMVs. The 4,346 samples were also detected using a reported RT-PCR assay, and the results showed this RT-PCR assay was less sensitive than the assays reported here. Of the 70 confirmed positives, 40 were class I Newcastle disease virus (NDV or APMV-1) and 27 were class II NDV from poultry including chickens, ducks, geese, and pigeons, and three were APMV-2 from parrots. The surveillance identified APMV-2 in parrots for the first time, and revealed that prevalence of NDVs in live poultry markets was higher than that in poultry farms. The surveillance also suggested that class I NDVs in chickens could be as prevalent as in ducks, and class II NDVs in ducks could be more prevalent than in chickens, and class II NDVs could be more prevalent than class I NDVs in ducks. Altogether, we developed a set of specific and sensitive RT-PCR assays for detection of all known APMVs, and conducted a large-scale surveillance using the assays which shed novel insights into APMV epidemiology.

Update on the large-scale screening of ALK fusion oncogene transcripts in archival NSCLC tumor specimens using multiplexed RT-PCR assays.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7594-7594 ◽  
Author(s):  
Tianhong Li ◽  
Eric Huang ◽  
Sonal Desai ◽  
Laurel Beckett ◽  
Craig Stephens ◽  
...  
Keyword(s):  
Large Scale ◽  
Fusion Gene ◽  
Standard Of Care ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts ◽  
Gene Transcripts ◽  
Pcr Assays ◽  
Alk Positive ◽  
Large Scale Screening

7594 Background: The ALK inhibitor crizotinib offers a new standard of care for advanced NSCLC patients with EML4-ALK fusion oncogenes. We previously reported a 4.0% frequency of EML4-ALK fusion oncogene transcripts detected in 1889 NSCLC specimens in the RGI database (Li et al., ASCO 2011). Methods: Patented single and multiplexed RT-PCR assays suitable for rapid and accurate detection of all variants of ALK fusion oncogene transcripts were used as previously described, including all 9 known EML4-ALK fusion gene transcripts and ALK RNA levels (Danenberg, ASCO 2010). The sensitivity and specificity on archival formalin-fixed, paraffin-embedded tumor specimens are 99% and 100%, respectively. We here update the detection of EML4-ALK fusion transcripts in the RGI database. Results: Between 12/2009 and 09/2011, 4750 NSCLC specimens in the RGI database were tested for the presence of ALK fusion transcripts. We found 152 (3.2%) NSCLC cases with EML4-ALK fusion positivity, including 87 (57.2%) V1, 15 (9.9%) V2, 47 (30.9%) V3, and 3 (2.0%) V5a variants. Median age (range): 61.1 (33-96). Female: 74 (49%). All EML4-ALK-positive tumors were adenocarcinomas. No EGFR or K-Ras mutation was detected in ALK fusion-positive samples. Expression of chemotherapy-related biomarkers was available from 63 (female: 31, 49%) EML4-ALK-positive cases in the database: 43 (68%) had low TS level of <2.33; 40 (63.5%) had low ERCC1 level of <1.7, and 25 (40%) had low RRM1 level of <0.97. Conclusions: This RT-PCR assay provides a tool for rapid, large-scale screening of NSCLC FFPE tissues for EML4-ALK fusion gene transcripts. The relative value of this RT-PCR assay as a companion diagnostic test for drugs targeting ALK merits evaluation in comparison with the FDA approved ALK FISH test.

scholarly journals A specific real-time PCR assay for the detection of Bordetella pertussis

2007 ◽  
Vol 56 (7) ◽  
pp. 918-920 ◽  
Author(s):  
Benoit Vincart ◽  
Ricardo De Mendonça ◽  
Sylvianne Rottiers ◽  
Françoise Vermeulen ◽  
Marc J. Struelens ◽  
...  
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Pcr Assays ◽  
Reference Strains ◽  
Respiratory Specimens

A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.

Comparative Evaluation of In Vitro and In Vivo Assays for the Detection of Avian Infectious Bronchitis Virus as a Contaminant of Live Poultry Vaccines

1998 ◽  
Vol 26 (5) ◽  
pp. 629-634
Author(s):  
Emiliana Falcone ◽  
Edoardo Vignolo ◽  
Livia Di Trani ◽  
Simona Puzelli ◽  
Maria Tollis
Keyword(s):  
Infectious Bronchitis Virus ◽  
Serological Response ◽  
Avian Infectious Bronchitis Virus ◽  
Animal Testing ◽  
Rt Pcr ◽  
Pcr Assay ◽  
In Vivo Test ◽  
Infectious Bronchitis

A reverse transcriptase polymerase chain reaction (RT-PCR) assay specific for identifying avian infectious bronchitis virus (IBV) in poultry vaccines, and the serological response to IBV induced by the inoculation of chicks with a Newcastle disease vaccine spiked with the Massachusetts strain of IBV, were compared for their ability to detect IBV as a contaminant of avian vaccines. The sensitivity of the IBV-RT-PCR assay provided results which were at least equivalent to the biological effect produced by the inoculation of chicks, allowing this assay to be considered a valid alternative to animal testing in the quality control of avian immunologicals. This procedure can easily be adapted to detect a number of contaminants for which the in vivo test still represents the only available method of detection.

scholarly journals High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening

2020 ◽  
Author(s):  
Rania Francis ◽  
Marion Le Bideau ◽  
Priscilla Jardot ◽  
Clio Grimaldier ◽  
Didier Raoult ◽  
...  
Keyword(s):  
High Speed ◽  
Large Scale ◽  
Vaccine Development ◽  
Work Load ◽  
Drug Susceptibility Testing ◽  
High Content Screening ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Positive Elements

AbstractSARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

scholarly journals Best Molecular Tools to Investigate Coronavirus Diversity in Mammals: A Comparison

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1975
Author(s):  
Petra Drzewnioková ◽  
Francesca Festa ◽  
Valentina Panzarin ◽  
Davide Lelli ◽  
Ana Moreno ◽  
...  
Keyword(s):  
Literature Review ◽  
In Silico ◽  
Host Shift ◽  
Molecular Tools ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Field Samples ◽  
Low Sensitivity

Coronaviruses (CoVs) are widespread and highly diversified in wildlife and domestic mammals and can emerge as zoonotic or epizootic pathogens and consequently host shift from these reservoirs, highlighting the importance of veterinary surveillance. All genera can be found in mammals, with α and β showing the highest frequency and diversification. The aims of this study were to review the literature for features of CoV surveillance in animals, to test widely used molecular protocols, and to identify the most effective one in terms of spectrum and sensitivity. We combined a literature review with analyses in silico and in vitro using viral strains and archive field samples. We found that most protocols defined as pan-coronavirus are strongly biased towards α- and β-CoVs and show medium-low sensitivity. The best results were observed using our new protocol, showing LoD 100 PFU/mL for SARS-CoV-2, 50 TCID50/mL for CaCoV, 0.39 TCID50/mL for BoCoV, and 9 ± 1 log2 ×10−5 HA for IBV. The protocol successfully confirmed the positivity for a broad range of CoVs in 30/30 field samples. Our study points out that pan-CoV surveillance in mammals could be strongly improved in sensitivity and spectrum and propose the application of a new RT-PCR assay, which is able to detect CoVs from all four genera, with an optimal sensitivity for α-, β-, and γ-.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

Inhibitors of histone deacetylases in class I and class II suppress human osteoclasts in vitro

2011 ◽  
Vol 226 (12) ◽  
pp. 3233-3241 ◽  
Author(s):  
M.D. Cantley ◽  
D.P. Fairlie ◽  
P.M. Bartold ◽  
K.D. Rainsford ◽  
G.T. Le ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Class Ii ◽  
Class I ◽  
Human Osteoclasts

296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Mohana Kumar ◽  
W. J. Lee ◽  
Y. M. Lee ◽  
R. Patil ◽  
S. L. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Class Ii ◽  
In Vitro Differentiation ◽  
Rt Pcr ◽  
Hepatic Differentiation ◽  
Alizarin Red ◽  
Differentiation Potential

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

scholarly journals Large Scale Jets in Class I and Class II Radio Sources and Quasars

1989 ◽  
Vol 8 ◽  
pp. 409-416
Author(s):  
G.V. Bicknell
Keyword(s):  
Interstellar Medium ◽  
Surface Brightness ◽  
Large Scale ◽  
External Medium ◽  
Class Ii ◽  
Class I ◽  
Radio Sources ◽  
Speed Of Light ◽  
Slow Surface ◽  
Quasar Jets

ABSTRACTThe physics of large scale jets in class I and class II extragalactic radio sources and quasars is discussed. Class I jets appear to be turbulent, transonic jets which entrain the interstellar medium. The related jet deceleration causes a slow surface brightness decline which is usually observed. Class II jets are supersonic and terminate in an advancing shock against the external medium. Both types of jet are initially light but the ratio of jet density to external density of class I jets increases owing to entrainment. It is quite plausible that quasar jets are hypersonic and light and this may solve problems of confinement. The velocities of class I jets are of the order of a few thousand kilometers per second. Class II and quasar jets may be at least mildly relativistic. However, it is not clear whether the velocities of large scale jets in powerful sources are close to the speed of light. Recent depolarization measurements provide an interesting focus for discussion of this question.

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