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-.

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.

Quantification of viable eggs of the potato cyst nematodes (Globodera spp.) using either trehalose or RNA-specific Real-Time PCR

Nematology ◽  
2014 ◽  
Vol 16 (10) ◽  
pp. 1219-1232 ◽  
Author(s):  
Johanna E. Beniers ◽  
Thomas H. Been ◽  
Odette Mendes ◽  
Marga P.E. van Gent-Pelzer ◽  
Theo A.J. van der Lee
Keyword(s):  
Real Time ◽  
Visual Inspection ◽  
Membrane Integrity ◽  
Globodera Pallida ◽  
Potato Cyst Nematodes ◽  
Original Sample ◽  
Rt Pcr ◽  
Cyst Nematodes ◽  
Pcr Assay ◽  
Field Samples

Two novel methods for the quantitative estimation of the number of viable eggs of the potato cyst nematodes (Globodera pallida and G. rostochiensis) were tested and compared with visual inspection. One is based on the loss of membrane integrity upon death and uses trehalose (a disaccharide) as a marker, the second test exploits the rapid degeneration of mRNA upon decease with a RNA-specific Real-Time Polymerase Chain Reaction (RT-PCR) assay. The viability of eggs in suspensions with different numbers of eggs was determined morphologically and was compared with both trehalose and elongation-factor-1-alpha (EF1α) mRNA measurements. The trehalose assay provided results that were close to those of the visual assessment using a microscope but only when samples contained low numbers of eggs. The lowest detectable value is 1.1 egg in the original sample and small differences in the number of viable eggs can be detected. Unfortunately, trehalose measurements reached a saturation limit at 1 cyst 10 μl−1; therefore, samples with nematode numbers above 262 eggs have to be diluted. The presence of dead cysts did not have a negative effect on the trehalose measurements. However, the use of egg suspensions instead of encysted eggs improved both the trehalose absorbance and the reliability of the measurements. When cysts were exposed to a treatment with allylisothiocyanate, the trehalose measurement detected the presence of more viable eggs than a hatching assay. The RT-PCR assay required a minimum of 30 eggs before detection occurred but can detect up to 8000 eggs in a 25 μl sample, which is an advantage when samples with high PCN infestations have to be processed. However, the confidence intervals (CI) of the RT-PCR assay are larger than those of the trehalose assay, which results in a high variation of single measurements. For example, at a density of 210 eggs in the original sample the 95% CI for the trehalose assay covers 191-228 eggs, and the 95% CI for the RT-PCR assay for G. pallida lies between 73 and 602 eggs and for G. rostochiensis between 59 and 745 eggs. Trials with field samples using both methods supported the laboratory tests. 95% of the field samples tested with the trehalose assay lie within the CI of the standard curve compared to 58% of the RT-PCR tested samples for G. pallida. The measurements of the field samples of G. pallida and G. rostochiensis populations using both methods resulted in larger numbers of viable eggs being detected compared to a hatching test. Neither of the investigated methods in their current state of development is optimal for use as a substitute for the visual inspection used in monitoring labs. The variance of the RT-PCR assay is too high if used for quantitative monitoring; the density range of eggs that can be detected using the trehalose assay is too small.

scholarly journals Evaluation of 2-Thioxoimadazolidin-4-One Derivatives as Potent Anti-Cancer Agents through Apoptosis Induction and Antioxidant Activation: In Vitro and In Vivo Approaches

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 83
Author(s):  
Mohamed S. Nafie ◽  
Ahmed I. Khodair ◽  
Hebat Allah Y. Hassan ◽  
Noha M. Abd El-Fadeal ◽  
Hanin A. Bogari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
In Silico ◽  
Hepg2 Cells ◽  
Flow Cytometric Analysis ◽  
Apoptosis Induction ◽  
Rt Pcr ◽  
Anti Cancer ◽  
Ic50 Values

Background: Hepatocellular carcinoma (HCC) is one of the most widespread malignancies and is reported as the fourth most prevalent cause of cancer deaths worldwide. Therefore, we aimed to investigate the probable mechanistic cytotoxic effect of the promising 2-thioxoimidazolidin-4-one derivative on liver cancer cells using in vitro and in vivo approaches. The compounds were tested for the in vitro cytotoxic activity using MTT assay, and the promising compound was tested in colony forming unit assay, flow cytometric analysis, RT-PCR, Western blotting, in vivo using SEC-carcinoma and in silico to highlight the virtual mechanism of action. Both compounds 4 and 2 performed cytotoxic effects against HepG2 cells with IC50 values of 0.017 and 0.18 μM, respectively, compared to Staurosporine and 5-Fu as reference drugs with IC50 values of 5.07 and 5.18 µM, respectively. Compound 4 treatment revealed apoptosis induction by 19.35-fold (11.42% compared to 0.59% in control), arresting the cell cycle at G2/M phase. Moreover, studying gene expression that plays critical roles in cell cycle and apoptosis by RT-PCR demonstrated that compound 4 enhances the expression of the pro-apoptotic genes p53, PUMA, and Caspase 3, 8, and 9, and impedes the anti-apoptotic Bcl-2 gene in the HepG2 cells. It can also inhibit the PI3K/AKT pathway at both gene and protein levels, which was reinforced by the in silico predictions of the molecular docking simulations towards the PI3K/AKT proteins. Finally, in vivo study verified that compound 4 has a promising anti-cancer activity through activating antioxidant levels (CAT, SOD and GSH) and ameliorating hematological, biochemical, and histopathological findings.

scholarly journals Detection of SARS-CoV-2 in saliva: implications for specimen transport and storage

2020 ◽  
Author(s):  
Eloise Williams ◽  
Nicole Isles ◽  
Brian Chong ◽  
Katherine Bond ◽  
Yano Yoga ◽  
...  
Keyword(s):  
N Gene ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Two Samples ◽  
Polymerase Chain ◽  
The Stability ◽  
Gamma Irradiated ◽  
And Storage ◽  
Time Point

Saliva has recently been proposed as a suitable specimen for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Use of saliva as a diagnostic specimen may present opportunities for SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) testing in remote and low-resource settings. Determining the stability of SARS-CoV-2 RNA in saliva over time is an important step in determining optimal storage and transport times. We undertook an in vitro study to assess whether SARS-CoV-2 could be detected in contrived saliva samples. The contrived saliva samples comprised 10 ml pooled saliva spiked with gamma-irradiated SARS-CoV-2 to achieve a concentration of 2.58×104 copies ml SARS-CoV-2, which was subsequently divided into 2 ml aliquots comprising: (i) neat saliva; and a 1 : 1 dilution with (ii) normal saline; (iii) viral transport media, and (iv) liquid Amies medium. Contrived samples were made in quadruplicate, with two samples of each stored at either: (i) room temperature or (ii) 4 °C. SARS-CoV-2 was detected in all SARS-CoV-2 spiked samples at time point 0, day 1, 3 and 7 at both storage temperatures using the N gene RT-PCR assay and time point 0, day 1 and day 7 using the Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, USA) RT-PCR assay. The ability to detect SARS-CoV-2 in saliva over a 1 week period is an important finding that presents further opportunities for saliva testing as a diagnostic specimen for the diagnosis of SARS-CoV-2.

scholarly journals Development and Use of a Reverse Transcription-PCR Assay To Study Expression of Tri5 byFusarium Species In Vitro and In Planta

1999 ◽  
Vol 65 (9) ◽  
pp. 3850-3854 ◽  
Author(s):  
F. M. Doohan ◽  
G. Weston ◽  
H. N. Rezanoor ◽  
D. W. Parry ◽  
P. Nicholson
Keyword(s):  
Reverse Transcription ◽  
Infected Plant ◽  
Fusarium Culmorum ◽  
Pcr Analysis ◽  
Growth Stages ◽  
In Planta ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Glucuronidase Activity

ABSTRACT The Tri5 gene encodes trichodiene synthase, which catalyzes the first reaction in the trichothecene biosynthetic pathway. In vitro, a direct relationship was observed between Tri5expression and the increase in deoxynivalenol production over time. We developed a reverse transcription (RT)-PCR assay to quantifyTri5 gene expression in trichothecene-producing strains ofFusarium species. We observed an increase inTri5 expression following treatment of Fusarium culmorum with fungicides, and we also observed an inverse relationship between Tri5 expression and biomass, as measured by β-d-glucuronidase activity, during colonization of wheat (cv. Avalon) seedlings by F. culmorum. RT-PCR analysis also showed that for ears of wheat cv. Avalon inoculated with F. culmorum, there were different levels of Tri5 expression in grain and chaff at later growth stages. We used the Tri5-specific primers to develop a PCR assay to detect trichothecene-producing Fusariumspecies in infected plant material.

Serological Evidence of Avian Metapneumovirus Infection in Layer Chicken Farms of Madhya Pradesh, India

2021 ◽  
Author(s):  
Manoj Kumar ◽  
Chakradhar Tosh ◽  
S. Nagarajan ◽  
Harshad V. Murugkar ◽  
Naveen Kumar ◽  
...  
Keyword(s):  
Control Strategies ◽  
Geographical Area ◽  
Madhya Pradesh ◽  
N Gene ◽  
Viral Agent ◽  
Upper Respiratory Tract ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Avian Metapneumovirus

Background: Avian metapneumovirus (aMPV) is an important viral agent in chicken and turkey production industry and is most commonly associated with acute upper respiratory tract infection and swollen head syndrome. The disease is of significant economic concern as it is highly contagious and can lead to production losses in chicken and turkey flocks, especially when associated with secondary bacterial pathogens. Methods: Herein, we have performed a survey on layer farms in selected areas of Madhya Pradesh State, India to ascertain the aMPV status in laying birds, choanal cleft swabs and sera samples were collected for ELISA and RT-PCR assay. A total of 263 sera and 169 choanal cleft swabs from five districts of Madhya Pradesh State (Indore, Bhopal, Sagar, Guna and Bhind) from layer birds of age group 20-72 weeks were screened for aMPV antibodies and for genome. Result: Out of these Indore had maximum sero-postivity (73.33%), followed by Sagar (5.00%) and Bhopal (4.08%), respectively. Other two districts did not show any seropostivity against aMPV. RT-PCR assay using published primers and in vitro transcribed RNA of nucleoprotein (N) gene as positive control was carried out in 169 choanal cleft samples from four districts (Indore, Bhopal, Sagar and Guna). All the samples were found negative for aMPV genome by RT-PCR assay. Overall, 26/263 (9.88%) sera were positive to aMPV antibodies. As the chickens in India are not vaccinated against avian metapneumovirus therefore, this study indicates that layers are exposed to this important poultry pathogen. This study warrants further investigation in wider geographical area and isolation of aMPV to design the control strategies for aMPV.

The survey of porcine teschoviruses in field samples in China with a universal rapid probe real-time RT-PCR assay

2012 ◽  
Vol 45 (4) ◽  
pp. 1057-1061 ◽  
Author(s):  
Chaofan Zhang ◽  
Zhongtian Wang ◽  
Feng Hu ◽  
Yebing Liu ◽  
Zheng Qiu ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Field Samples

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.

scholarly journals The Level of Effectiveness Use of Quinoline Drugs in COVID-19: A Literature Review

2020 ◽  
Vol 4 (2) ◽  
pp. 151
Author(s):  
M.Dedi Dermawan Dermawan ◽  
Afrita Amalia Laitupa ◽  
Muslim Andala Putra ◽  
Nenny Triastuti
Keyword(s):  
Literature Review ◽  
Lupus Erythematosus ◽  
Rt Pcr ◽  
Group Rate ◽  
Enzymatic Activation ◽  
Ct Test ◽  
Ph Increase ◽  
Test Result ◽  
Anti Inflammation

ABSTRACT          Chloroquine is the first line of medicine in the treatment of malaria. Besides being antimalaria, the chloroquine also can be used as the anti-inflammation in the medicine of arthritis rheumatoid arthritis and lupus erythematosus discoid. Hydroxychloroquine sulfate is 4-aminoquinolin with hydroxylated chloroquine analog, having the same pharmacokinetic as chloroquine which is given orally in hydroxychloroquine sulfate form, processed by gastrointestinal absorption and very faster kidney elimination. The effectiveness of chloroquine and hydroxychloroquine towards COVID-19 in the in vitro experiment showed it could inhibit the duplication of the SARS-CoV-2 virus. The chloroquine function is to stop COVID-19 infection with (EC50) 1,13 μM and (CC50) larger than 100 μM. Meanwhile, the hydroxychloroquine function is to inhibit the attachment and entry of the virus into the host’s cell by enzymatic activation which is the lysosome acidification disorder and antigen presentation as the result of pH increase. Based on the clinical study, the 10 of 12 patients who have lopinavir/ritonavir therapy by virology, the chloroquine group showed RT-PCR negative on day 7, 10, and 14 in compare to lopinavir/ritonavir that showed RT-PCR negative on day 14. On the 9th day, 60% of the patients of chloroquine group showed the CT scan of Lungs image normal instead of the lopinavir/ritonavir at 25%. In the day 14 based on the CT test result, the pulmonary improvement increased twice rather than chloroquine group (Rate Ratio 2.21). It proved that the chloroquine role showed the result of the medicine has a significant effect by cleaning the virus or other clinical matters. The purpose of this literature review is to know the effectiveness quinoline class of drugs which is chloroquine and hydroxychloroquine in COVID-19 disease.Keywords: effectiveness, chloroquine, COVID-19Correspondence: [email protected]

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