RT-PCR

Canine Distemper Virus detection based in Hemaglutinine Gene as target in Reverse Transcriptase-Polymerase Chain Reaction

Published on: 27th December, 2018

OCLC Number/Unique Identifier: 7964755439

Canine Distemper have become a major concern within the veterinary clinical work. Thus, the appearance of many cases of canine distemper in adult animals with their vaccination plan up to date has alarmed veterinarians. Many cases of canine distemper have become a major concern within the clinical veterinary task. The purpose of this work was to detect the gene of hemagglutinin of the virus canine distemper, by using the Polymerase Chain Reaction associated with Reverse transcription (RT-PCR), as confirmation of the clinical diagnosis of disease. For this, peripheral blood samples of animals were used clinically patients and were grouped according to their date of extraction and using vaccines commercial as control. The results allow to demonstrate a high sensitivity of the technique, besides allowing the use of samples up to seven days of storage at 4°C, despite the fragility of RNA viral. Detection of canine distemper virus haemagglutinin gene in field samples and its high sensitivity, suggests studying its use as a diagnostic tool complementary to the clinical diagnosis of canine distemper in our country.
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Use of the Reverse Transcription-Polymerase Chain Reaction for differential detection of two lineages of the canine distemper virus in Chile

Published on: 4th March, 2019

OCLC Number/Unique Identifier: 8056345040

Worldwide, Canine Distemper Virus (CDV) infection is a highly prevalent disease with high morbidity and mortality. CDV causes a multisystemic disease in a wide range of hosts including 9 families of mammals among them some primates, cetaceans and numerous carnivores. It presents a high tropism for lymphoid, neurological and epithelial tissue, leading to an infection of almost all systems, so the clinical signs observed are very varied. The diagnosis is made based on the clinical presentation of the disease, which considers a variety of signs and must be confirmed by a laboratory diagnostic method. The molecular technique called Reverse Transcription Polymerase Chain Reaction (RT-PCR) has been used to characterize viral strains based on the basis of genetic differences on the hemagglutinin (H) gene of CDV has allowed the identification of 14 circulating lineages in the world. Two lineages, namely the America-1 and the Europe-1/South America-1 have been described in Chile. The goal of this work was to implement a multiplex RT-PCR protocol, which was built on the in silico design of primers based on the H gene nucleotide sequences stored in the Genbank® database. This method was capable of detecting the previously described two circulating genetic lineages of CDV in a differential way providing a supporting diagnostic tool for epidemiological studies in the country. These results suggest that the primers described here are extremely selective for the above-mentioned lineages. In addition, our initial screening indicated that most analyzed clinical samples corresponded to the America-1 lineage, stressing the need for a continuous surveillance in order to properly address the prevalence of both lineages in Chile.
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Role of HRCT Thorax in preoperative assessment of RT-PCR COVID-19 negative oral cancer patients

Published on: 22nd December, 2020

OCLC Number/Unique Identifier: 8872656815

Background: Oral cancer accounts for 30% of all types of cancer in India. Surgery is the mainstay of treatment but due to the recent outbreak of COVID-19, there is a partial or complete disruption of health services in the country. The initial delay in the treatment was due to cancellations of planned surgeries as per government regulations and reduction in public transportation. In the latter half of the initial relaxation of the lockdown, we formulated our institutional protocol for the surgical treatment of oral cancer patients. On admission, all patients were kept in isolated wards followed by RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) testing on the same day. RT-PCR negative patients are subjected to HRCT Thorax (High-resolution Computer Tomography). This ensured the safety of health care workers, patients, and patient attendees. Keeping this in mind, we did an observational study on the role of HRCT in the pre-operative screening of asymptomatic oral cancer patients.  Materials and methods: Retrospective analysis of prospectively collected data of 150 patients was done. HRCT reporting was done by two experienced senior radiologists of the Department of Radiology at our hospital. Results: The number of patients under the CO-RADS 1 category was 121 and CO-RADS 2 category was 29, according to CO-RADS classification. The results of RT-PCR and HRCT were compared and there was a 100% positive correlation between RT-PCR and HRCT Thorax. Conclusion: Our study supported the use of HRCT Thorax as a diagnostic tool in pre-operative screening of oral cancer patients for COVID 19, particularly in RT-PCR negative cases.
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Sequence-independent single-primer-amplification (SISPA) as a screening technique for detecting unexpected RNA viral adventitious agents in cell cultures

Published on: 12th March, 2021

OCLC Number/Unique Identifier: 8982622827

The sequence-independent, single-primer amplification (SISPA) enables the random amplification of nucleic acids, allowing the detection and genome sequencing of different viral agents. This feature of SISPA method provides evidence for application of it in monitoring the presence of adventitious RNA viruses in cell cultures. We evaluated SISPA method for the detection of a challenge RNA virus representing adventitious agent in cell cultures. Besides, by optimizing the SISPA method in our laboratory, we found false-positive results on negative control lanes in electrophoresis gels. To investigate the sources of contamination, false-positive results of SISPA were cloned into Escherichia coli cells, sequenced, and phylogenetically analyzed. This data revealed that the SISPA method can be used as an adjunct method to confirm the absence of unexpected adventitious RNA viruses in cell cultures. The phylogenetic analysis of SISPA contaminant sequences showed that the false-positive results were caused by nucleic acid amplification of commercial cDNA synthesis kit reagents, probably tracing back to expression plasmids and host ribosomal sequences, used for the production of enzymes. Therefore, laboratories using random amplification methods must be constantly aware of the potentials of such contaminations, yielding false-positive results and background noise in the final NGS reads.
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Pigeonpea sterility mosaic virus a green plague-Current status of available drug and new potential targets

Published on: 14th June, 2021

OCLC Number/Unique Identifier: 9124811236

Pigeonpea is one of the important legume crops with high protein content and nutritional traits. It has enormous potency for its widespread adoption by farming communities. It is affected by various kinds of biotic and abiotic stresses. In the context, of biotic stresses Sterility mosaic disease (SMD) is one of the severe diseases in pigeonpea which ultimately lead to the drastic yield loss. The virus belongs to the genus Emaravirus, family- Fimoviridae. SMD is associated with two diverse types of Emaravirus, Pigeonpea sterility mosaic virus1 (PPSMV-1) and Pigeonpea sterility mosaic virus 2 (PPSMV-2). It is transmitted by the mite (Aceria cajani), mainly environmental contributing to the feasibility for the mites for the inoculation of the virus. The SMD is mainly governed by two genes SV1 that includes the dominant allele and serves as an inhibitory action on the resistance of the SV2. Methods for identification of the virus include RT-PCR, DIBA and ELISA using alkaline phosphatase or penicillinase. To control SMV disease farmers generally adopted intercropping methods. There are few potential drugs have been identified for the administration of the disease such as 0.1% Fenazaquin, Dicofol, Imidacloripid, Carbosulfan; Spiromesifin includes the inhibition of the mite inoculation on the pigeonpea plant. The present review describes compressive and systematic insights on SMV protein targets and potential drugs that could be utilized as the presumed drug targets for the finding of true drugs against the SMD in pigeonpea.
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COVID-19 disease with persistently negative RT-PCR test for SARS-CoV-2

Published on: 7th August, 2020

OCLC Number/Unique Identifier: 8653632638

Introduction: The disease outbreak of COVID-19 has had a great clinical and microbiological impact in the last few months. In the preanalytical phase, the collection a sample from of a respiratory tract at the adequate moment and from the correct anatomical site is essential for a rapid and precise molecular diagnosis with a false negative rate of less than 20%. Materials and methods: We conducted a descriptive study of COVID-19 disease with a persistently negative RT-PCR test in patients seen at the National Institute of Respiratory Diseases (INER) in Mexico City in the period of March through May of 2020. 38 patients were registered with negative RT-PCR test obtained through nasopharyngeal and oropharyngeal swabbing. We evaluated the distribution of data with the Shapiro-Wilk test of normality. The non-parametric data are reported with median. The nominal and ordinal variables are presented as percentages. Results: The average age of our cohort was 46 years and 52.63% were male (n = 20). Diabetes Mellitus was documented in 34.21% (n = 13) of the patients, Systemic Hypertension in 21.05% (n = 8), Obesity in 31.57% (n = 12) and Overweight in 42.10% (n = 16). Exposure to tobacco smoke was reported in 47.36% (n = 18) of the patients. The median initial saturation of oxygen was 87% at room air. The severity of the disease on admission was: mild 71.05% (n = 27), moderate 21.05% (n = 8) and severe or critical in 7.89% (n = 3) of the cases respectively. 63.15% (n = 24) sought medical care after 6 or more days with symptoms. Lymphopenia was documented in 78.94% (n = 30). Median LDH at the time of admission was 300, being elevated in 63.15% (n = 24) of the cases. The initial tomographic imaging of the chest revealed predominantly ground glass pattern in 81.57% (n = 31) and predominantly consolidation in 18.42% (n = 7). The registered mortality was 15.78% (n = 6). Conclusion: Patients with COVID-19 and a persistently negative RT-PCR test with fatal outcomes did not differ from the rest of the COVID-19 population since they present with the same risk factors shared by the rest of patients like lymphopenia, comorbidities, elevation of D-Dimer and DHL on admission as well as a tomographic COVID-19 score of severe illness, however we could suggest that the percentage of patients with a mild form of the disease is higher in those with a persistently negative RT-PCR test.
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Comparison of clinical, chest CT and laboratory findings of suspected COVID-19 inpatients with positive and negative RT-PCR

Published on: 15th February, 2021

OCLC Number/Unique Identifier: 9038776103

Introduction: COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 and it was first reported in China. The aim of this study was to compare clinical features, chest CT findings and laboratory examinations of suspected COVID-19 inpatients according to RT-PCR analysis. Methods: Demographics, comorbidites, symptoms and signs, laboratory results and chest CT findings were compared between positive and negative groups. The study included 292 patients (134 females, 158 males) suspected of COVID-19. All statistical calculations were performed with SPSS 23.0. Results: 158 (54.1%) of the cases were male and 134 (45.9%) were female. Their ages ranged from 17 to 95 years, with an average of 50.46 ± 20.87. A symptom or sign was detected in 86.3% of all patients. The chest CT images of 278 patients were analyzed. Chest CT was negative in 59.2% of patients with positive RT-PCR and 43.9% of patients with negative RT-PCR results. Chest CT findings were atypical or indeterminate in 22.4% of patients with positive RT-PCR results and 20% of patients with negative RT-PCR analysis. ALP, bilirubine, CRP, eosinophil count, glucose, CK-MB mass and lactate were significantly lower in patients with positive RT-PCR test. LDH, lipase, MCV, monocyte, neutrophil count, NLR, platelet, pO2, pro-BNP, procalcitonin, INR, prothrombin time, sodium, troponin T, urea, WBC were significantly lower in patients with positive RT-PCR test results. Conclusion: The diagnosis of COVID-19 is based on history of patient, typical symptoms or clinical findings. Chest CT, RT-PCR and laboratory abnormalities make the diagnosis of disease stronger.
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Expression of C-type Natriuretic Peptide and its Specific Guanylyl Cyclase-Coupled Receptor in Pig Ovarian Granulosa Cells

Published on: 22nd August, 2018

OCLC Number/Unique Identifier: 7844580667

Background: C-type natriuretic peptide (CNP) was isolated from porcine brain and is a 22-amino acid peptide which belongs to the natriuretic peptide (NP) family. Even though this peptide shares structural similarity to other endogenous NPs including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) its receptor selectivity is different from other NPs. The present study was undertaken to investigate the expression of C-type natriuretic peptide (CNP) and its specific guanylyl cyclase (GC)-coupled receptor in the granulosa cells of the pig ovarian follicle. Results: Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the granulosa cell layer of the ovarian follicle with an apparent dissociation constant (Kd>) and a maximal binding capacity (Bmax) of 1.41±0.39 nM and 2.75±0.65 fmol/mm2 respectively. Binding of 125I-[Tyr0]-CNP(1-22) to these sites was also prevented by atrial natriuretic peptide (ANP(1-28)), brain natriuretic peptide (BNP(1-26)) and des[Gln18,Ser19,Gly20, Leu21,Gly22] ANP(4-23) (C-ANP). Production of 3’,5’-cyclic guanosine monophosphate (cGMP) by particulate GC in the granulosa cell membranes was stimulated by natriuretic peptides (NPs) with a rank order of potency of CNP(1-22)>>BNP(1-26)>ANP(1-28). HS-142-1, a selective antagonist of the two recognized GC-coupled NPRs, inhibited CNP(1-22)-stimulated cGMP production in granulosa cell membranes in a dose-dependent manner. Also mRNAs for all three recognized NPRs were detected in granulosa cells using reverse transcriptase-polymerase chain reaction (RT-PCR). Serial dilution curves of granulosa cell extracts were parallel to the standard curve of synthetic CNP. Conclusion: These results indicate that CNP and its specific receptor are expressed in the granulosa cells of the pig ovary, and suggest that CNP may be a local autocrine and/or paracrine regulator via activation of its specific GC-coupled receptor, NPR-B.
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Cloning and Characterization of a Pseudo-Response Regulator 7 (PRR7) Gene from Medicago Sativa Involved In Regulating the Circadian Clock

Published on: 16th July, 2019

OCLC Number/Unique Identifier: 8185507893

The circadian clock is an endogenous molecular oscillator with a period of about 24 hours, which regulates the physiology and developmental processes of almost all higher plants. Pseudo-response regulators (PRRs) are an important part of the central clock oscillator, together with other clock genes, constituting interlinked transcriptional feedback loops, which partly influence plant growth and development. In this study, a circadian clock-related gene MsPRR7 was cloned from Medicago sativa (alfalfa) by homologous cloning. The full length MsPRR7 gene was 2648 bp in length, with an open reading frame of 2385 bp encoding a protein of 795amino acids. Phylogenetic analysis showed that the MsPRR7 was closely related to PRR7 from the PRR family of Arabidopsis thaliana. Subcellular localization analysis found that MsPRR7 was located in the nucleus. Quantitative reverse-transcription polymerase chain reactions (qRT-PCR) demonstrated that expression of MsPRR7 gene transcripts in leaves was affected by circadian rhythms, and that its expression level increased with an extension of illumination time, reaching a peak around 8–10 hours. These results will provide the experimental basis for further study of the regulation of PRR family genes in alfalfa.
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Serological and molecular characterization of two seed born cowpea mosaic Comovirus isolates affecting cowpea plants (Vigna unguiculata L.) in northern Egypt

Published on: 1st October, 2019

OCLC Number/Unique Identifier: 8282686936

Cowpea plants naturally infected with cowpea mosaic comovirus (CPMV) showed different mosaic, mottle, dwarfing, and vain clearing symptoms. Diseased plants were ollected from certain locations of Alexandria and El-Beheira governorates during the growing seasons from 2011 to 2012. CPMV was detected in infected sap at 8 to 24 days after inoculation by DBIA, indirect ELISA and tissue blot immunoassay (TBIA). Chlorotic local lesions were observed on Chenopodium amaranticolor in infectivity test. By using indirect ELISA and DBIA, CPMV were detected in infected plant sap of serial dilutions up to 1: 400. The incidence of CPMV in 21 day old cowpea seedlings grown from infected seeds was determined by ELISA and positive detection of virus antigen reached 65%. Nitrocellulose membrane and canson paper could be used as solid carriers in TBIA and DBIA for detection of CPMV in infected plant tissues. Results revealed that both faces of nitrocellulose membrane and canson paper could be used as solid carriers in TBIA for detection of CPMV in infected plant tissues. According to reverse transcription polymerase chain reaction (RT-PCR) assay of CPMV infected plant; the amplified product was approximately 800bp of partial coat protein gene. The nucleotide sequences accession number were LN606585 and LN606586. The phylogenetic tree was generated using sequences of CPMV isolates with the other CPMV records from GenBank.
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Artemisia Naphta: A novel oil extract for sensitive and acne prone skin

Published on: 15th June, 2021

OCLC Number/Unique Identifier: 9124793207

Background: The plant Artemisia annua has been used in traditional Chinese medicine for many years. Rich in bioactive molecules, the A. annua plant is used to extract the anti-malaria compound artemisinin (< 1%), which results in most of the plant being unutilized. One byproduct of artemisinin extraction is artemisia naphtha (AN), which has yet to be studied extensively. Aims: Study the activity of a novel AN oil extract against microbes, pro-inflammatory cytokines, and dermatological endpoints that are key for eczema and acne pathogenesis to determine if an effective A. annua extract for these skin conditions can be developed. Methods: Gas chromatography-mass spectrometry was performed to determine the composition of AN oil. P. acnes, S. aureus, M. furfur, and C. albicans were cultured to determine minimal inhibitory concentration. in vitro studies utilizing keratinocytes and macrophages were treated with AN oil and gene expression measured by quantitative RT-PCR. A 13-subject clinical trial was performed with 1% AN oil Gel to assess its potential benefits for sensitive and acne prone skin. Results: AN oil upregulates filaggrin gene expression and possesses antimicrobial and anti-inflammatory activity inhibiting LPS, S. aureus and "Th2 induced" pro-inflammatory mediator release (IL-6, IL-8 and TSLP). Clinical assessment of 1% AN Gel shows it reduces acne blemishes and the appearance of redness. Conclusion: Previously an underutilized and unpurified byproduct, AN is now the source to develop the first topical AN oil for cosmetic use with an activity profile that suggests it is effective for those with sensitive and/or acne prone skin.
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Evaluation of the LumiraDx SARS-CoV-2 antigen assay for large-scale population testing in Senegal

Published on: 5th January, 2022

OCLC Number/Unique Identifier: 9391752258

Purpose: Real-time reverse-transcription polymerase chain reaction (RT-PCR)-based testing remains the gold standard for the diagnosis of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the high diagnosis demand of SARS-CoV-2 and the limited resources for RT-PCR testing, especially in Low-Income Countries (LICs), antigen-based methods are being considered as an option. The aim of this study was to assess the performance of LumiraDx SARS-CoV-2 antigen assay for large population screening compared to RT-PCR.Methods: This evaluation was conducted on 4146 participants including travelers and participants under household survey and vaccine evaluation studies before injection of the first dose. Oropharyngeal and nasopharyngeal swaps were collected from each participant into 2 mL of viral transport medium (VTM) and 400 μl of VTM were used to assess the performance of LumiraDx SARS-CoV-2 antigen assay, compared to RT-PCR. Results: The prevalence of SARS-CoV-2 of the cohort was 4.5% with RT-PCR and 4.1% with LumiraDx antigen test. Compared to the RT-PCR, the sensitivity and specificity of the LumiraDx antigen SARS-CoV-2 test were 82,7% [95% CI 74.1-89,7] and 99.9% [95% CI 99.6-99.9] respectively. Given the RT-PCR threshold cycle (Ct) range, the sensitivity was 92.1% [95% CI 84.6-96.3] when the Ct value was below or equal 33 cycles, and 38.1% [95% CI 18.9-61.3] when it was above 33 cycles. The inter-rater reliability showed a kappa coefficient of 0.88 when considering all the patients and 0.94 for Ct values below 33 cycles. Conclusion: Our data have shown that the LumiraDx platform can be considered for large-scale testing of SARS-CoV-2.
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Applications of convolutional neural networks in chest X-ray analyses for the detection of COVID-19

Published on: 20th January, 2022

OCLC Number/Unique Identifier: 9391752257

Throughout global efforts to defend against the spread of COVID-19 from late 2019 up until now, one of the most crucial factors that has helped combat the pandemic is the development of various screening methods to detect the presence of COVID-19 as conveniently and accurately as possible. One of such methods is the utilization of chest X-Rays (CXRs) to detect anomalies that are concurrent with a patient infected with COVID-19. While yielding results much faster than the traditional RT-PCR test, CXRs tend to be less accurate. Realizing this issue, in our research, we investigated the applications of computer vision in order to better detect COVID-19 from CXRs. Coupled with an extensive image database of CXRs of healthy patients, patients with non-COVID-19 induced pneumonia, and patients positive with COVID-19, convolutional neural networks (CNNs) prove to possess the ability to easily and accurately identify whether or not a patient is infected with COVID-19 in a matter of seconds. Borrowing and adjusting the architectures of three well-tested CNNs: VGG-16, ResNet50, and MobileNetV2, we performed transfer learning and trained three of our own models, then compared and contrasted their differing precisions, accuracies, and efficiencies in correctly labeling patients with and without COVID-19. In the end, all of our models were able to accurately categorize at least 94% of the CXRs, with some performing better than the others; these differences in performance were largely due to the contrasting architectures each of our models borrowed from the three respective CNNs.
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