Reverse Transcription-polymerase Chain Reaction Method Research Articles

Targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for malate-aspartate shuttle and tumour progression.

A series of studies have demonstrated the emerging involvement of transfer RNA (tRNA) processing during the progression of tumours. Nevertheless, the roles and regulating mechanisms of tRNA processing genes in neuroblastoma (NB), the prevalent malignant tumour outside the brain in children, are yet unknown. Analysis of multi-omics results was conducted to identify crucial regulators of downstream tRNA processing genes. Co-immunoprecipitation and mass spectrometry methods were utilised to measure interaction between proteins. The impact of transcriptional regulators on expression of downstream genes was measured by dual-luciferase reporter, chromatin immunoprecipitation, western blotting and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods. Studies have been conducted to reveal impact and mechanisms of transcriptional regulators on biological processes of NB. Survival differences were analysed using the log-rank test. c-Myc was identified as a transcription factor driving tRNA processing gene expression and subsequent malate-aspartate shuttle (MAS) in NB cells. Mechanistically, c-Myc directly promoted the expression of glutamyl-prolyl-tRNA synthetase (EPRS) and leucyl-tRNA synthetase (LARS), resulting in translational up-regulation of glutamic-oxaloacetic transaminase 1 (GOT1) as well as malate dehydrogenase 1 (MDH1) via inhibiting general control nonrepressed 2 or activating mechanistic target of rapamycin signalling. Meanwhile, lamin A (LMNA) inhibited c-Myc transactivation via physical interaction, leading to suppression of MAS, aerobic glycolysis, tumourigenesis and aggressiveness. Pre-clinically, lobeline was discovered as a LMNA-binding compound to facilitate its interaction with c-Myc, which inhibited aminoacyl-tRNA synthetase expression, MAS and tumour progression of NB, as well as growth of organoid derived from c-Myc knock-in mice. Low levels of LMNA or elevated expression of c-Myc, EPRS, LARS, GOT1 or MDH1 were linked to a worse outcome and a shorter survival time of clinical NB patients. These results suggest that targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for MAS and tumour progression.

COVID-19 Hierarchical Classification Using a Deep Learning Multi-Modal.

Coronavirus disease 2019 (COVID-19), originating in China, has rapidly spread worldwide. Physicians must examine infected patients and make timely decisions to isolate them. However, completing these processes is difficult due to limited time and availability of expert radiologists, as well as limitations of the reverse-transcription polymerase chain reaction (RT-PCR) method. Deep learning, a sophisticated machine learning technique, leverages radiological imaging modalities for disease diagnosis and image classification tasks. Previous research on COVID-19 classification has encountered several limitations, including binary classification methods, single-feature modalities, small public datasets, and reliance on CT diagnostic processes. Additionally, studies have often utilized a flat structure, disregarding the hierarchical structure of pneumonia classification. This study aims to overcome these limitations by identifying pneumonia caused by COVID-19, distinguishing it from other types of pneumonia and healthy lungs using chest X-ray (CXR) images and related tabular medical data, and demonstrate the value of incorporating tabular medical data in achieving more accurate diagnoses. Resnet-based and VGG-based pre-trained convolutional neural network (CNN) models were employed to extract features, which were then combined using early fusion for the classification of eight distinct classes. We leveraged the hierarchal structure of pneumonia classification within our approach to achieve improved classification outcomes. Since an imbalanced dataset is common in this field, a variety of versions of generative adversarial networks (GANs) were used to generate synthetic data. The proposed approach tested in our private datasets of 4523 patients achieved a macro-avg F1-score of 95.9% and an F1-score of 87.5% for COVID-19 identification using a Resnet-based structure. In conclusion, in this study, we were able to create an accurate deep learning multi-modal to diagnose COVID-19 and differentiate it from other kinds of pneumonia and normal lungs, which will enhance the radiological diagnostic process.

Rapid and sensitive visual detection of avian leukosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow immunochromatographic strip assay.

Considering that avian leukosis virus (ALV) infection has inflicted massive economic losses on the poultry breeding industry in most countries, its early diagnosis remains an important measure for timely treatment and control of the disease, for which a rapid and sensitive point-of-care test is required. We established a user-friendly, economical, and rapid visualization method for ALV amplification products based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with an immunochromatographic strip in a lateral flow device (LFD). Using the ALVp27 gene as the target, five RT-LAMP primers and one fluorescein-isothiocyanate-labeled probe were designed. After 60 min of RT-LAMP amplification at 64 °C, the products could be visualized directly using the LFD. The detection limit of this assay for ALV detection was 102 RNA copies/μL, and the sensitivity was 100 times that of reverse transcription polymerase chain reaction (RT-PCR), showing high specificity and sensitivity. To verify the clinical practicality of this assay for detecting ALV, the gold standard RT-PCR method was used for comparison, and consistent results were obtained with both assays. Thus, the assay described here can be used for rapid detection of ALV in resource-limited environments.

Basic biochemical and hematological parameters in COVID-19 deceased: Analysis of potential mortality predictors

Introduction: As a result of research conducted globally, experts continue to extensively study the long-term consequences that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections can have on patients, as well as the factors contributing to severe outcomes. The aim of this study was to investigate the values of basic biochemical and hematological parameters in patients with fatal outcomes of SARS-CoV-2 infection, as well as to determine the combination of hematological and biochemical parameters that contribute to the progression of SARS-CoV-2 infection. The examined parameters were correlated with the age and gender distribution of patients with fatal outcomes from the SARS-CoV-2 infection. Methods: The study represents a retrospective study of patients hospitalized at the Cantonal Hospital Zenica from February to April 2021, focusing on the biochemical and hematological parameters of subjects with confirmed presence of the SARS-CoV-2 virus using the reverse transcription-polymerase chain reaction methods who were hospitalized at the Cantonal Hospital Zenica. Results: Of the 250 deceased subjects in the sample, females comprised the relative majority at 53.6%. Among the examined parameters, hemoglobin, mean corpuscular hemoglobin (MCH), and MCH were significantly lower in females compared to males, while males had significantly higher values of urea, creatinine, and troponin. The average age of the patients was 74 years, and with aging, the values of erythrocytes, hemoglobin, and troponin increased. Troponin showed a statistically significant positive correlation with age, as well as with urea and creatinine. Conclusion: We can conclude that hemoglobin, urea, creatinine, and troponin are parameters that can be considered to contribute to the progression of SARS-CoV-2 infection. These parameters can be useful for assessing disease severity and prognosis in patients infected with the SARS-CoV-2 virus.

Wing expansion functional analysis of ion transport peptide gene in Sogatella furcifera (Horváth) (Hemiptera: Delphacidae)

Ion transport peptide (ITP), a superfamily of arthropod neuropeptides, serves a crucial role in regulating various physiological processes such as diuresis, ecdysis behavior, and wing expansion. However, the molecular characteristics, expression profile, and role of ITP in Sogatella furcifera are poorly understood. To elucidate the characteristics and biological function of ITP in S. furcifera, we employed reverse transcription-polymerase chain reaction (RT-PCR) and RNA interference (RNAi) methods. The identified SfITP gene encodes 117 amino acids. The expression of SfITP gradually increased followed the formation of 3-day-old of 5th instar nymph, peaking initially at 40 min after eclosion, and reaching another peak 24 h after eclosion, with particularly high expression levels in thorax and wing tissues. Notably, SfITP RNAi in 3rd instar nymphs of S. furcifera significantly inhibited the transcript levels of SfITP, resulting in 55% mortality and 78% wing deformity. These findings suggests that SfITP is involved in the regulation of wing expansion in S. furcifera, providing insights into the regulation of insect wing expansion and contributing to the molecular understanding of this process.

Occult bacteremia in living donor liver transplantation: a prospective observational study of recipients and donors.

To investigate the incidence and clinical impact of occult bacteremia in liver transplantation (LT). This prospective observational study involved a fixed-point observation for up to 2weeks after living donor LT in 20 recipients, with 20 donors as comparison subjects. Bacteria in the blood samples were detected using the ribosomal RNA-targeted reverse-transcription quantitative polymerase chain reaction method. To identify the causality with the gut microbiota (GM), fecal samples were collected and analyzed simultaneously. Occult bacteremia was identified in four recipients (20%) and three donors (15%) before the operation, and in seven recipients (35%) and five donors (25%) after the operation. Clostridium leptum subgroup, Prevotella, Colinesella, Enterobacteriaceae, and Streptococcus were the main pathogens responsible. Although it did not negatively affect the donor post-hepatectomy outcomes, the recipients with occult bacteremia had a higher rate of infectious complications post-LT. The GM analyses showed fewer post-LT predominant obligate anaerobes in both the recipients and donors with occult bacteremia. Occult bacteremia is a common condition that occurs in both donors and recipients. While occult bacteremia generally remains subclinical in the healthy population, there is potential risk of the development of an apparent post-LT infection in recipients who are highly immunosuppressed.

Detection of Foot and Mouth Disease Virus in Cattle in Lamongan and Surabaya, Indonesia Using RT-PCR Method

Foot and mouth disease (FMD) virus was reported as an outbreak in Indonesia in April 2022 and belonged to serotype O/ME-SA/Ind-2001e is spread in the country. This study aimed to detect the causative agent based on clinical symptoms in cattle that have the vesicle in the mouth and hooves. A total of 25 samples were collected during August 2022 from Lamongan and Surabaya, Indonesia. FMD was identified in 58% (7/12) using reverse transcription polymerase chain reaction (RT-PCR), respectively. The samples were performed using universal primers with 328 bp length for primary diagnosis of FMD. These findings indicate that the spread of FMD viruses is highly contagious, so rapid and accurate diagnosis is needed as an effort to control and monitor FMD viruses.

MicroRNA-30a inhibits cell proliferation in a sepsis-induced acute kidney injury model by targeting the YAP-TEAD complex

BackgroundAcute kidney injury (AKI) is a primary feature of renal complications in patients with sepsis. MicroRNA (miRNA/miR)-30a is an essential regulator of cardiovascular diseases, tumors, phagocytosis, and other physical processes, but whether it participates in sepsis-induced AKI (sepsis-AKI) is unknown. We aimed to elucidate the functions and molecular mechanism underlying miR-30a activity in sepsis-AKI. MethodsThe classical cecal ligation and puncture (CLP) method and lipopolysaccharide (LPS)-induced Human Kidney 2 (HK-2) cells were used to establish in vivo and in vitro sepsis-AKI models. Specific pathogen-free and mature male Sprague-Dawley (SD) rats, aged 6–8 weeks (weight 200–250 g), were randomly divided into five-time phase subgroups. Fluid resuscitation with 30 mL/kg 37 °C saline was administered after the operation, without antibiotics. Formalin-fixed, paraffin-embedded kidney sections were stained with hematoxylin and eosin. SD rat kidney tissue samples were collected for analysis by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. HK-2 cells were transfected with hsa-miR-30a-3p mimics or inhibitors, and compared with untreated normal controls. RNA, protein, and cell viability were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot, and cell counting kit-8 methods. A Dual-Luciferase Assay Kit (Promega) was used to measure luciferase activity 48 h after transfection with miR-30a-3p mimics. ResultsExpression levels of miR-30a-3p and miR-30a-5p in renal tissues of the sepsis group were significantly reduced at 12 h and 24 h (P <0.05). Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly increased in renal tissue 3 h after the operation in rats (P <0.05), and gradually decreased 6 h, 12 h, and 24 h after CLP. Levels of miR-30a-5p and miR-30a-3p were significantly down-regulated at 3 h after LPS treatment (P <0.05), and gradually decreased in HK-2 cells. One hour after LPS (10 µg/mL) treatment, TNF-α and IL-1β levels in HK-2 cells were significantly up-regulated (P < 0.05), and they were markedly down-regulated after 3 h (P <0.05). IL-6 expression levels began to rise after LPS treatment of cells, peaked at 6 h (P <0.05), and then decreased to the initial level within a few hours. Stimulation with 10 µg/mL LPS promoted HK-2 cells proliferation, which was inhibited after miR-30a-3p-mimic transfection. Bioinformatics prediction identified 37 potential miR-30a-3p target genes, including transcriptional enhanced associate domain 1 (TEAD1). After transfection of HK-2 cells with miR-30a-3p mimics and miR-30a-3p inhibitor, TEAD1 transcript was significantly up- and down-regulated, respectively (both P <0.05). After LPS treatment (24 h), expression of TEAD1 in the inhibitors group was significantly increased (P <0.01), while that in the mimics group was significantly suppressed (P <0.01). In the dual luciferase reporter experiment, miR-30a-3p overexpression decreased fluorescence intensity (P <0.01) from TEAD1-wt-containing plasmids, but did not influence fluorescence intensity from TEAD1-muta-containing plasmids. LPS may promote HK-2 cells proliferation through the miR-30a-3p/TEAD1 pathway. ConclusionIn a background of expression of inflammatory factors, including TNF-α, IL-1β, and IL-6, which were transiently increased in the sepsis-AKI model, miR-30a was down-regulated. Down-regulated miR-30a-3p may promote cell proliferation by targeting TEAD1 in LPS-induced HK-2 cells, demonstrating its potential as a biomarker for early sepsis-AKI diagnosis.

Imipenem resistance associated with amino acid alterations of the OprD porin in Pseudomonas aeruginosa clinical isolates.

Globally, the spread of carbapenem-resistant strains has limited treatment options for multidrug-resistant (MDR) Pseudomonas aeruginosa infections. This study aimed to determine the role of point mutations as well as the expression level of the oprD gene in the emergence of imipenem-resistant P.aeruginosa strains isolated from patients referred to Ardabil hospitals. A total of 48 imipenem-resistant clinical isolates of P. aeruginosa collected between June 2019 and January 2022 were used in this study. Detection of the oprD gene and its amino acid alterations was performed using the polymerase chain reaction (PCR) and DNA sequencing techniques. The expression level of the oprD gene in imipenem-resistant strains was determined using the real-time quantitative reverse transcription PCR (RT-PCR) method. All imipenem-resistant P. aeruginosa strains were positive for the oprD gene based on the PCR results, and also five selected isolates indicated one or more amino acid alterations. Detected amino acid alterations in the OprD porin were Ala210Ile, Gln202Glu, Ala189Val, Ala186Pro, Leu170Phe, Leu127Val, Thr115Lys, and Ser103Thr. Based on the RT-PCR results, the oprD gene was downregulated in 79.1% of imipenem-resistant P. aeruginosa strains. However, 20.9% of strains showed overexpression of the oprD gene. Probably, resistance to imipenem in these strains is associated with the presence of carbapenemases, AmpC cephalosporinase, or efflux pumps. Owing to the high prevalence of imipenem-resistant P. aeruginosa strains due to various resistance mechanisms in Ardabil hospitals, the implementation of surveillance programs to reduce the spread of these resistant microorganisms along with rational selection and prescription of antibiotics is recommended.

Role of acetyl-CoA acetyltransferase 1 expression in the molecular mechanism of adenomyosis.

Adenomyosis is a benign uterine illness characterized by endometrial gland and stromal invasion into the myometrium. Acetyl-CoA acetyltransferase 1 (ACAT1) is an enzyme localized in mitochondria that is involved in ketogenesis and ketolysis processes by reversibly catalyzing the formation of acetoacetyl-CoA from two acetyl-CoA molecules. The current study investigated the expression of the ACAT1 molecule in tissue samples of patients diagnosed with adenomyosis and healthy endometrial tissues. It is aimed to determine the differences in ACAT1 gene expression and in this way to discover the first information about the role of ACAT1 in the development and molecular mechanism of adenomyosis. In the current retrospective study, formalin-fixed paraffin-embedded archival tissues were employed. A total of 76 patient samples were included in the study. Of these samples, 28 are adenomyotic tissue (Group I), 30 are eutopic endometrial tissue (Group II), and 18 are the Control Group. In these groups, the expression levels of the ACAT1 gene were determined by the reverse transcription-polymerase chain reaction method. When the expression results of the ACAT1 gene were evaluated, statistically significant differences were found between the groups (p<0.05). There was a difference between Group I-Group II and Group I-Control Group regarding the ACAT1 gene. No statistically significant change was observed between Group II and Control Group. It is a remarkable finding that the expression of ACAT1 in adenomyosis tissue is decreased compared with both eutopic endometrium and control groups tissues. The results suggest that ACAT1 may be associated with the molecular pathogenesis of adenomyosis.

A strategy for entomo-virological surveillance of yellow fever, dengue, zika, and chikungunya viruses in field-collected mosquitoes

Arboviruses transmitted by Culicidae insects are significant threats to human health, presenting dynamic transmission cycles and involving different vectors and hosts. The surveillance and characterization of the vectors involved in these cycles are crucial for understanding and preventing potential outbreaks. Therefore, we propose a strategy that we used for entomological surveillance of urban, rural, and sylvatic mosquitoes and to characterize natural infection by four major arboviruses.•Immature and adult mosquitoes were collected intra, peri and extradomicilie of urban and rural households, using different collection methodologies.•Mosquitoes were pooled or separated in head-thorax and abdomen, according to the species.•A multiplex nested RT-PCR (Reverse transcription polymerase chain reaction) method was used for the simultaneous detection of dengue virus (DENV), zika virus (ZIKV), chikungunya virus (CHIKV), and yellow fever virus (YFV).Overall, this strategy proved helpful for vectors surveillance at different ecosystems, as well as for implementing a low-cost molecular surveillance system that allows the early detection of potential outbreaks, and identify other potential vectors involved in viral transmission.

The impact of COVID-19 pandemic on surveillance of influenza and influenza-like viruses: a single center experience

Aims: Influenza and the agents responsible for influenza-like illness (ILI) are a significant cause of upper and lower respiratory tract infections, with a notable pattern of seasonal incidence. The surveillance of influenza and agents causing ILI is important for the developmentof a prophylaxis approach and infection control. In the present study, we utilize the Reverse transcription-polymerase chain reaction (RT-PCR) method to identify the presence of influenza virus and other agents associated with ILI in nasopharyngeal smears obtained from patients exhibiting symptoms of influenza and ILI. The study sample included patients who were admitted to multiple clinics and intensive care units (ICU) of the Health Sciences University Ankara Training and Research Hospital in the period preceding, and in the initial months and first year following the coronavirus disease 2019 (COVID-19) outbreak. Throughthe comparisons made in the study, the intention is to gain a better understanding of the influence of the COVID-19 pandemic on other viral infectious agents transmitted via the respiratory tract.&#x0D; Methods: Included in the study were 257 admitted to different wards and ICUs of our hospital due to symptoms of upper or lower respiratory tract infection and ILI between 2015 and 2021 (excluding data from 2017–2018). The study was conducted prospectively within the scope of the Global Influenza Hospital Surveillance Network project. Using sterile swabs, nasopharyngeal swab samples were collected from inpatients who provided informed consent for their participation in the study, and the samples were placed in a viral transport medium. The presence of influenza viruses (Influenza A, subtypes of Influenza A and Influenza B) and agents causing ILI (i.e. respiratory syncytial virus, coronavirus) was investigated with an RT-PCR approach.&#x0D; Results: The most common symptoms among the 257 patients included in the study with pre-diagnoses of influenza and ILI were cough (82.2%), fever (67.7%), shortness of breath (66.1%) and myalgia (40%). The RT-PCR detected a viral agent in 60 (23.3%) of the 257 patients, whereas no agent could be detected in 197 (76.6%) patients. Furthermore, 51 (18.5%) tested positive for influenza virus, five (1.9%) for respiratory syncytial virus (RSV), and four (1.5%) for SARS-CoV-2. An analysis of the results within two distinct time frames, namely prior to the COVID-19 pandemic (2016–2017, 2019–prior to December 10, 2020) and during the COVID-19 pandemic (between December 11, 2020 and 2021), influenza viruses (influenza A H1N1 and influenza A H3N2), RSV and influenza type B were identified as the dominant viruses before the COVID-19 pandemic, while the predominant viruses were a single influenza strain and four SARS-CoV-2 variants during the COVID-19 pandemic.A significant difference was noted in the distribution of viruses between the two time frames – prior to the pandemic and during the pandemic. Of the patients, 199 (77.4%) were discharged with full recovery while 58 patients died (22.6%). Of the 58 patients that died, 25 were female (25/131 females) and 33 were male (33/126 males), 11 had tested positive for influenza virus andone for RSV, while no infectious agent could be identified in 46 patients. &#x0D; Conclusion: The implementation of molecular testing methods for the identification of viral infectious agents among inpatients during influenza and ILI outbreaks, administering antiviral and prophylactic treatments targeting influenza, RSV and SARS-CoV-2 infections, and adopting infection control measures, could significantly decrease mortality and morbidity rates while mitigating the complications associated with these infectious agents.

MicroRNA expression profile of human umbilical vein endothelial cells in response to coxsackievirus A10 infection reveals a potential role of miR-143-3p in maintaining the integrity of the blood-brain barrier.

Coxsackievirus A10 (CV-A10) has been one of the main etiologies of hand, foot, and mouth disease (HFMD) epidemics in recent years and can cause mild to severe illness and even death. Most of these severe and fatal cases were closely associated with neurological impairments, but the potential mechanism of neuropathological injury triggered by CV-A10 infection has not been elucidated. MicroRNAs (miRNAs), implicated in the regulation of gene expression in a post-transcriptional manner, play a vital role in the pathogenesis of various central nervous system (CNS) diseases; therefore, they serve as diagnostic biomarkers and are emerging as novel therapeutic targets for CNS injuries. To gain insights into the CV-A10-induced regulation of host miRNA-processing machinery, we employed high-throughput sequencing to identify differentially expressed miRNAs in CV-A10-infected human umbilical vein endothelial cells (HUVECs) and further analyzed the potential functions of these miRNAs during CV-A10 infection. The results showed that CV-A10 infection could induce 189 and 302 significantly differentially expressed miRNAs in HUVECs at 24 and 72 hpi, respectively, compared with the uninfected control. Moreover, the expression of four selected miRNAs and their relevant mRNAs was determined to verify the sequencing data by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) methods. After that, gene target prediction and functional annotation revealed that the targets of these dysregulated miRNAs were mostly enriched in cell proliferation, signal transduction, cAMP signalling pathway, cellular response to interleukin-6, ventral spinal cord interneuron differentiation, negative regulation of glial cell differentiation, neuron migration, positive regulation of neuron projection development, etc., which were primarily involved in the processes of basic physiology, host immunity, and neurological impairments and further reflected vital regulatory roles of miRNA in viral pathogenicity. Finally, the construction of a miRNA-regulated network also suggested that the complex regulatory mechanisms mediated by miRNAs might be involved in viral pathogenesis and virus-host interactions during CV-A10 infection. Furthermore, among these dysregulated miRNAs, miR-143-3p was demonstrated to be involved in the maintenance of blood-brain barrier (BBB) integrity.

COVID-19 scent dog research highlights and synthesis during the pandemic of December 2019-April 2023.

This review was undertaken to provide information concerning the advancement of research in the area of COVID-19 screening and testing during the worldwide pandemic from December 2019 through April 2023. In this review, we have examined the safety, effectiveness, and practicality of utilizing trained scent dogs in clinical and public situations for COVID-19 screening. Specifically, results of 29 trained scent dog screening peer-reviewed studies were compared with results of real-time reverse-transcription polymerase chain reaction (RT-PCR) and rapid antigen (RAG) COVID-19 testing methods. The review aims to systematically evaluate the strengths and weaknesses of utilizing trained scent dogs in COVID-19 screening. At the time of submission of our earlier review paper in August 2021, we found only four peer-reviewed COVID-19 scent dog papers: three clinical research studies and one preprint perspective paper. In March and April 2023, the first author conducted new literature searches of the MEDLINE/PubMed, Google Scholar, and Cochrane Library websites. Again, the keyword phrases utilized for the searches included "COVID detection dogs," "COVID scent dogs," and "COVID sniffer dogs." The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 Checklist was followed to ensure that our review adhered to evidence-based guidelines for reporting. Utilizing the results of the reviewed papers, we compiled statistics to intercompare and summarize basic information concerning the scent dogs and their training, the populations of the study participants, the types of sampling methods, the comparative tests utilized, and the effectiveness of the scent dog screening. A total of 8,043 references were identified through our literature search. After removal of duplicates, there were 7,843 references that were screened. Of these, 100 were considered for full-text eligibility, 43 were included for qualitative synthesis, and 29 were utilized for quantitative analysis. The most relevant peer-reviewed COVID-19 scent dog references were identified and categorized. Utilizing all of the scent dog results provided for this review, we found that 92.3 % of the studies reached sensitivities exceeding 80 and 32.0 % of the studies exceeding specificities of 97 %. However, 84.0 % of the studies reported specificities above 90 %. Highlights demonstrating the effectiveness of the scent dogs include: (1) samples of breath, saliva, trachea-bronchial secretions and urine as well as face masks and articles of clothing can be utilized; (2) trained COVID-19 scent dogs can detect presymptomatic and asymptomatic patients; (3) scent dogs can detect new SARS-CoV-2 variants and Long COVID-19; and (4) scent dogs can differentiate SARS-CoV-2 infections from infections with other novel respiratory viruses. The effectiveness of the trained scent dog method is comparable to or in some cases superior to the real-time RT-PCR test and the RAG test. Trained scent dogs can be effectively utilized to provide quick (seconds to minutes), nonintrusive, and accurate results in public settings and thus reduce the spread of the COVID-19 virus or other viruses. Finally, scent dog research as described in this paper can serve to increase the medical community's and public's knowledge and acceptance of medical scent dogs as major contributors to global efforts to fight diseases.

A New Method to Detect Variants of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Bioluminescent Assay in Real Time (RT-LAMP-BART)

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100–200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500–3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern.

Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China.

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5'-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

Evaluation of droplet digital qRT-PCR (dd qRT-PCR) for quantification of SARS CoV-2 RNA in stool and urine specimens of COVID-19 patients.

There have been a few reports of viral load detection in stool and urine samples of patients with coronavirus disease 2019 (COVID-19), and the transmission of the virus through faecal oral route. For clinical diagnosis and treatment, the widely used reverse transcription-polymerase chain reaction (qRT-PCR) method has some limitations. The aim of our study to assess the presence and concentration of SARS CoV-2 RNA in stool and urine samples from COVID-19 patients with mild, moderate, and severe disease, we compared a traditional qRT-PCR approach with a ddPCR. ddPCR and qRT-PCR-based target gene analysis were performed on 107 COVID-19-confirmed patients paired samples (N1 and N2). The MagMax magnetic beads base method was used to isolate RNA. Real-time qRT-PCR and dd PCR were performed on all patients. The average cycle threshold (Ct) of qRT-PCR was highly correlated with the average copy number of 327.10 copies/l analyzed in ddPCR. In ddPCR, urine samples showed 27.1% positivity while for stool it was 100%. This study's findings not only show that SARS CoV-2 is present in urine and faeces, but also suggest that low concentrations of the viral target ddPCR make it easier to identify positive samples and help resolve for cases of inconclusive diagnosis.

Vaccination status for mild and asymptomatic infections with SARS-CoV-2 Omicron BA.2 variant in Shanghai.

This study aimed to evaluate the effects of different vaccine regimens on mild and asymptomatic infections with SARS-CoV-2 Omicron BA.2 variant in Shanghai. All asymptomatic patients and those with mild symptoms of Omicron infections were recruited from three major Fangcang shelter hospitals between March 26, 2022 and May 20, 2022. Nucleic acid for SARS-CoV-2 by real-time reverse-transcription polymerase chain reaction methods in nasopharyngeal swabs was assessed every day during the hospitalization. The value of cycle threshold lower than 35 was considered as positive result of SARS-CoV-2. A total of 214 592 cases were included in this study. The proportion of the asymptomatic patients was 76.90% and 23.10% of the recruited patients had mild symptoms. The median (interquartile range [IQR]: 25-75) duration of viral shedding (DVS) was 7 (5-10) days among all participants. The DVS varied greatly among different age groups. Children and the elderly had longer DVS compared with the adults. The booster shot of inactivated vaccine contributed to the shorter DVS in patients aged ≥70 years compared with the unvaccinated patients (8 [6-11] vs. 9 [6-12] days, p = 0.002]. Full inactivated vaccine regimen contributed to the shorter DVS in patients aged 3-6 years (7 [5-9] vs. 8 [5-10] days, p = 0.001]. In conclusion, the full inactivated vaccine regimen on children aged 3-6 years and booster inactivated vaccine regimen on the elderly aged ≥70 years appeared to be effective in reducing DVS. The booster vaccine regimen should be rigorously promoted and implemented.

Influence of functional-assembled-FGL polypeptide timbering material on expression of glial cell line-derived neurotropic growth factor mRNA in damaged rat’s spinal cord

Spinal cord injury is one of most serious complications when there is spine injury in patients. Spinal cord injury clinically involves anesthesia and mobility loss in leg in most of patients. Rats were herein molded and divided into groups, and Basso-Beattie-Bresnahan (BBB) method was adopted to assess the motor function of rats. The quantities of cassyl aspartic acid-specified protease (Caspase-3), neurofilament protein-200 (NF-200) and Glial Fibrillary Acidic Protein (GFAP)-positive cell were detected by immunohistochemical staining method. The expression of Glial cell line-derived neurotropic growth factor (GDNF) mRNA in spinal cord tissue was detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. The quantity of Caspase-3 positive cell and area of glial scar in the fiber grating laser-National Security (FGL-NS) group were significantly reduced compared with those from the other two groups. The quantities of NF-200 positive cell, graded result of Basso-Beattie-Bresnahan method and content of GDNF mRNA in the FGL-NS group were increased compared to those from the other two groups. The FGL-NS improved the expression of GDNF mRNA so as to repair the damaged neuronal cells.

Examination of the utility of the COVID-19 detection kit, TRC Ready® SARS-CoV-2 i for nasopharyngeal swabs.

The reverse transcription polymerase chain reaction (RT-PCR) offers high sensitivity, but has some drawbacks, such as the time required for the RNA extraction. Transcription reverse-transcription concerted reaction (TRC) Ready® SARS-CoV-2 i is easy to use and can be performed in about 40 minutes. TRC Ready® SARS-CoV-2 i and real-time one-step RT-PCR using the TaqMan probe tests of cryopreserved nasopharyngeal swab samples from patients diagnosed with COVID-19 were compared. The primary objective was to examine the positive and negative concordance rates. A total of 69 samples cryopreserved at -80° C were examined. Of the 37 frozen samples that were expected to be RT-PCR positive, 35 were positive by the RT-PCR method. TRC Ready® SARS-CoV-2 i detected 33 positive cases and 2 negative cases. One frozen sample that was expected to be RT-PCR positive was negative on both TRC Ready® SARS-CoV-2 i and RT-PCR. In addition, one frozen sample that was expected to be RT-PCR positive was positive by the RT-PCR method and negative by TRC Ready® SARS-CoV-2 i. Of the 32 frozen samples that were expected to be RT-PCR negative, both the RT-PCR method and TRC Ready® SARS-CoV-2 i yielded negative results for all 32 samples. Compared with RT-PCR, TRC Ready® SARS-CoV-2 i had a positive concordance rate of 94.3% and a negative concordance rate of 97.1%. TRC Ready® SARS-CoV-2 i can be utilized in a wide range of medical sites such as clinics and community hospitals due to its ease of operability, and is expected to be useful in infection control.