1,593 results match your criteria two-step pcr

Proliferation of SARS-CoV-2 B.1.1.7 Variant in Pakistan-A Short Surveillance Account.

Front Public Health 2021;9:683378. Epub 2021 May 31.

Department of Virology, National Institute of Health, Islamabad, Pakistan.

The emergence of a more transmissible variant of SARS-CoV-2 (B1. 1.7) in the United Kingdom (UK) during late 2020 has raised major public health concerns. Read More

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An Economical and Flexible Dual Barcoding, Two-Step PCR Approach for Highly Multiplexed Amplicon Sequencing.

Front Microbiol 2021 20;12:669776. Epub 2021 May 20.

Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna, Vienna, Austria.

In microbiome research, phylogenetic and functional marker gene amplicon sequencing is the most commonly-used community profiling approach. Consequently, a plethora of protocols for the preparation and multiplexing of samples for amplicon sequencing have been developed. Here, we present two economical high-throughput gene amplification and sequencing workflows that are implemented as standard operating procedures at the Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna. Read More

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A new molecular method for rapid etiological diagnosis of sepsis with improved performance.

Future Microbiol 2021 Jun 4. Epub 2021 Jun 4.

Microbiology & Virology Department, Azienda per l'Assistenza Sanitaria n 5 Friuli Occidentale, Via Montereale 24, Pordenone, 33170, Italy.

The value of blood cultures for confirming the clinical diagnosis of sepsis is suboptimal. There is growing interest in the potential of real-time PCR technology by detection of minute amounts of pathogen DNA in patient blood samples with results available within 4-6 h. Adopting a two-step approach, we evaluated the compliance of two versions of the MicrobScan assay on a total of 748 patients with suspected bloodstream infections. Read More

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Characterising Foot-and-Mouth Disease Virus in Clinical Samples Using Nanopore Sequencing.

Front Vet Sci 2021 17;8:656256. Epub 2021 May 17.

Vesicular Disease Reference Laboratory, The Pirbright Institute, Woking, United Kingdom.

The sequencing of viral genomes provides important data for the prevention and control of foot-and-mouth disease (FMD) outbreaks. Sequence data can be used for strain identification, outbreak tracing, and aiding the selection of the most appropriate vaccine for the circulating strains. At present, sequencing of FMD virus (FMDV) relies upon the time-consuming transport of samples to well-resourced laboratories. Read More

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A Markerless Gene Deletion System in by Using the Copper-Inducible YoeB Toxin as a Counterselectable Marker.

Microorganisms 2021 May 19;9(5). Epub 2021 May 19.

College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.

is an important zoonotic pathogen causing severe infections in swine and humans. Induction of the YoeB toxin in resulted in cell death, leading to the speculation that YoeB can be a counterselectable marker. Herein, the counterselection potential of YoeB was assessed in . Read More

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First Description of SARS-CoV-2 Infection in Two Feral American Mink () Caught in the Wild.

Animals (Basel) 2021 May 16;11(5). Epub 2021 May 16.

Department of Pharmacy, Facultad de CC de la Salud, UCH-CEU University, C/Ramon y Cajal s/n, 46113 Valencia, Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, is considered a pathogen of animal origin that is mainly transmitted from human to human. Several animal species can be naturally or experimentally infected by SARS-CoV-2, with compelling evidence that mink is highly susceptible to SARS-CoV-2 infection. Human-to-mink infection cases have been reported and there are also suggestions that mink-to-human infection occurs. Read More

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Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples.

Anal Chem 2021 06 25;93(22):7797-7807. Epub 2021 May 25.

Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801-3028, United States.

The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. Read More

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A novel strategy for SARS-CoV-2 mass screening with quantitative antigen testing of saliva: a diagnostic accuracy study.

Lancet Microbe 2021 May 19. Epub 2021 May 19.

International Medical Department, Hokkaido University Hospital, Sapporo, Japan.

Background: Quantitative RT-PCR (RT-qPCR) of nasopharyngeal swab (NPS) samples for SARS-CoV-2 detection requires medical personnel and is time consuming, and thus is poorly suited to mass screening. In June, 2020, a chemiluminescent enzyme immunoassay (CLEIA; Lumipulse G SARS-CoV-2 Ag kit, Fujirebio, Tokyo, Japan) was developed that can detect SARS-CoV-2 nucleoproteins in NPS or saliva samples within 35 min. In this study, we assessed the utility of CLEIA in mass SARS-CoV-2 screening. Read More

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ChromInst: A single cell sequencing technique to accomplish pre-implantation comprehensive chromosomal screening overnight.

PLoS One 2021 20;16(5):e0251971. Epub 2021 May 20.

Department of Research and Development, Yikon Genomics (Suzhou) Company Limited, Suzhou, Jiangsu, PR China.

Next Generation Sequencing (NGS) is a powerful tool getting into the field of clinical examination. Its preliminary application in pre-implantation comprehensive chromosomal screening (PCCS) of assisted reproduction (test-tube baby) has shown encouraging outcomes that improves the success rate of in vitro fertilization. However, the conventional NGS library construction is time consuming. Read More

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Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

ACS Nano 2021 May 19. Epub 2021 May 19.

Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. Read More

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Synthesis, Biological Evaluation, and QSAR Studies of 3-Iodochromone Derivatives as Potential Fungicides.

Front Chem 2021 30;9:636882. Epub 2021 Apr 30.

Division of Agricultural Chemicals, ICAR-Indian Agricultural Research Institute, New Delhi, India.

Despite the emergence of novel biotechnological and biological solutions, agrochemicals continue to play an important role in crop protection. Fungicide resistance is becoming a major problem; numerous cases of fungicide resistance have occurred worldwide in the last decade, resulting in the loss of several fungicides. The discovery of new molecules has therefore assumed critical importance in crop protection. Read More

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[A rapid and accurate method for herpesviral gnome editing].

Sheng Wu Gong Cheng Xue Bao 2021 Apr;37(4):1376-1384

College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, Henan, China.

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Read More

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Antibiotic exposure and the risk of hospital-acquired diarrhoea and Clostridioides difficile infection: a cohort study.

J Antimicrob Chemother 2021 May 10. Epub 2021 May 10.

Division of Epidemiology and Preventive Medicine, Tel Aviv Sourasky Medical Center, Israel.

Background: Hospital-acquired diarrhoea (HAD) and Clostridioides difficile infection (CDI) may be triggered by antibiotic use.

Objectives: To determine the effect of specific antibiotic agents and duration of therapy on the risk of HAD and CDI.

Patients And Methods: A single-centre retrospective cohort study was conducted between May 2012 and December 2014 in the internal medicine division. Read More

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Expression and significance of estrogen receptor and progesterone receptor in endometrial tissue of patients with intrauterine adhesions.

Gland Surg 2021 Apr;10(4):1478-1486

Department of Gynecology, The First People's Hospital of Yunnan Province and Affiliated Hospital of Kunming University of Science and Technology, Kunming, China.

Background: Our study aims to analyze the expression and significance of estrogen receptor (ER) and progesterone receptor (PR) in endometrial tissues of patients with intrauterine adhesion (IUA).

Methods: Fifty-four patients with IUAs examined in our hospital from January 2017 to January 2019 were selected as the research object (observation group), 54 healthy women who had physical examinations during the same period were selected as the control group, and the immunohistochemical EnVision was used. Two-step and real-time fluorescent quantitative PCR methods were used to detect the expression levels of ER and PR in the endometrial tissues of the two groups of subjects. Read More

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Rapid molecular syndromic testing for aetiological diagnosis of gastrointestinal infections and targeted antimicrobial prescription: experience from a reference paediatric hospital in Spain.

Eur J Clin Microbiol Infect Dis 2021 May 8. Epub 2021 May 8.

Institut de Recerca Sant Joan de Deu, Barcelona, Spain.

Aetiological diagnosis of gastrointestinal infections is challenging since a wide range of bacteria, parasites and viruses can be causal agents and derived clinical manifestations appear quite similar. Our aim was to evaluate contribution of the novel QIAstat-DxGastrointestinal Panel (GIP) to aetiological diagnosis of gastrointestinal infections and rational antimicrobial prescription in a reference paediatric hospital. Evaluation included comparison of diagnostic yield and agreement of results of QIAstat-Dx GIP and conventional microbiological methods. Read More

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Excellent option for mass testing during the SARS-CoV-2 pandemic: painless self-collection and direct RT-qPCR.

Virol J 2021 05 4;18(1):95. Epub 2021 May 4.

Institute of Applied Biotechnologies a.s., Prague, Czech Republic.

The early identification of asymptomatic yet infectious cases is vital to curb the 2019 coronavirus (COVID-19) pandemic and to control the disease in the post-pandemic era. In this paper, we propose a fast, inexpensive and high-throughput approach using painless nasal-swab self-collection followed by direct RT-qPCR for the sensitive PCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This approach was validated in a large prospective cohort study of 1038 subjects, analysed simultaneously using (1) nasopharyngeal swabs obtained with the assistance of healthcare personnel and analysed by classic two-step RT-qPCR on RNA isolates and (2) nasal swabs obtained by self-collection and analysed with direct RT-qPCR. Read More

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A two-step PCR assembly for construction of gene variants across large mutational distances.

Biol Methods Protoc 2021 2;6(1):bpab007. Epub 2021 Apr 2.

Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, West Bengal, India.

Construction of empirical fitness landscapes has transformed our understanding of genotype-phenotype relationships across genes. However, most empirical fitness landscapes have been constrained to the local genotype neighbourhood of a gene primarily due to our limited ability to systematically construct genotypes that differ by a large number of mutations. Although a few methods have been proposed in the literature, these techniques are complex owing to several steps of construction or contain a large number of amplification cycles that increase chances of non-specific mutations. Read More

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Induction of Plant Resistance in Tobacco against Tomato Spotted Wilt Orthotospovirus through Foliar Application of dsRNA.

Viruses 2021 04 12;13(4). Epub 2021 Apr 12.

Department of Plant Pathology, Washington State University, Pullman, WA 99163, USA.

Thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) continues to be a constraint to peanut, pepper, tobacco, and tomato production in Georgia and elsewhere. TSWV is being managed by an integrated disease management strategy that includes a combination of cultural practices, vector management, and growing virus-resistant varieties where available. We used a non-transgenic strategy to induce RNA interference (RNAi)-mediated resistance in tobacco () plants against TSWV. Read More

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First Report of Barley virus G in the United States and California.

Plant Dis 2021 Apr 27. Epub 2021 Apr 27.

University of California Davis, 8789, Plant Pathology, Davis, California, United States;

Barley (Hordeum vulgare) is a valuable annual cereal crop grown widely throughout the United States and the world. The majority of barley grown commercially in California and throughout the U.S. Read More

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Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification.

J Vis Exp 2021 03 31(169). Epub 2021 Mar 31.

Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences;

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. Read More

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Efficacy and safety of neoadjuvant therapy for HER2-positive early breast cancer: a network meta-analysis.

Ther Adv Med Oncol 2021 3;13:17588359211006948. Epub 2021 Apr 3.

Department of Breast Surgery, Fujian Medical University Union Hospital, No.29, Xin Quan Road, Gulou District, Fuzhou, Fujian Province, 350001, China.

Aims: Currently, there are many approaches available for neoadjuvant therapy for human epidermal growth factor receptor 2 (HER2)-positive breast cancer that improve therapeutic efficacy but are also controversial. We conducted a two-step Bayesian network meta-analysis (NMA) to compare odds ratios (ORs) for pathologic complete response (PCR) and safety endpoints.

Methods: The Cochrane Central Register of Controlled Trials, PubMed, Embase, and online abstracts from the American Society of Clinical Oncology and San Antonio Breast Cancer Symposium were searched comprehensively and systematically. Read More

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Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization.

PLoS One 2021 8;16(4):e0248581. Epub 2021 Apr 8.

Department of Food, Nutrition, and Bioprocessing Sciences, North Carolina State University, Raleigh, NC, United States of America.

Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. Read More

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Comparative Expression Analysis of Inflammatory and Immune-related Genes in Cattle During Acute Infection with Foot-and-mouth Disease Virus in Egypt.

J Vet Res 2021 Mar 26;65(1):39-44. Epub 2021 Jan 26.

Animal Behavior, Animal Husbandry and Animal Wealth Development Department, Faculty of Veterinary Medicine, Alexandria University, Alexandria Egypt.

Introduction: Foot-and-mouth disease is a highly infectious viral disease affecting all cloven-footed domestic animals. The three foot-and-mouth disease virus (FMDV) serotypes A, O and SAT2 are at present the greatest threat to susceptible animals in Egypt. The aim of the present study was, for the host factors associated with different FMDV infections in cattle during the acute phase, to compare these factors' influence on the expression of the , , , , , and inflammatory and immune-related genes. Read More

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The development of miniSTRs as a method for high-speed direct PCR.

Electrophoresis 2021 Apr 3. Epub 2021 Apr 3.

Department of Chemistry, Florida International University (FIU), Miami, Florida, USA.

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Read More

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Persistence of pv. in Field Soil in Central Europe.

Microorganisms 2021 Mar 13;9(3). Epub 2021 Mar 13.

Mendeleum-Institute of Genetics, Mendel University in Brno, Valticka 334, 691 44 Lednice, Czech Republic.

pv. () is a bacterium that causes black rot of crucifers. The greatest losses of brassica crop production usually result from seed-borne infection, but carry-over of inoculum in field soil may also be possible. Read More

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Gene Expression Analysis by Reverse Transcription Quantitative PCR.

Methods Mol Biol 2021 ;2283:61-74

Department of Applied Sciences, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland.

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid detection technology that allows the amplification and quantification of specific RNA transcripts. RT-qPCR has increasingly been adopted for the detection and quantification of H. pylori across a range of sample types and applications. Read More

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A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR.

APMIS 2021 Mar 17. Epub 2021 Mar 17.

Division of Clinical Neurology, Nuffield Department of Clinical Neurosciences, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. Read More

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Development of a two-step nucleic acid amplification test for accurate diagnosis of the Mycobacterium tuberculosis complex.

Sci Rep 2021 Mar 11;11(1):5750. Epub 2021 Mar 11.

Graduate Institute of Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan.

The Mycobacterium tuberculosis complex (MTBC) remains one of the top 10 leading causes of death globally. The early diagnosis of MTBC can reduce mortality and mitigate disease transmission. However, current nucleic acid amplification diagnostic test methods are generally time-consuming and show suboptimal diagnostic performance, especially in extrapulmonary MTBC samples or acid-fast stain (AFS)-negative cases. Read More

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Identification of Nontuberculous Mycobacteria in Patients with Pulmonary Diseases in Gyeongnam, Korea, Using Multiplex PCR and Multigene Sequence-Based Analysis.

Can J Infect Dis Med Microbiol 2021 22;2021:8844306. Epub 2021 Feb 22.

Department of Microbiology, College of Medicine, Gyeongsang National University, Jinju 52727, Republic of Korea.

Background: Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. Read More

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February 2021

IS26 cannot move alone.

J Antimicrob Chemother 2021 May;76(6):1428-1432

School of Life and Environmental Sciences, The University of Sydney, NSW 2006, Australia.

Background: IS26 plays a major role in the dissemination of antibiotic resistance determinants in Gram-negative bacteria.

Objectives: To determine whether insertion sequence IS26 is able to move alone (simple transposition) or if it exclusively forms cointegrates.

Methods: A two-step PCR using outward-facing primers was used to search for circular IS26 molecules. Read More

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