Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

A new approach for improved RT-PCR is described. It is based on primers designed to form controlled stem–loop and homodimer configurations, hence the name ‘double-bubble’ primers. The primers contain three main regions for efficient RT-PCR: a 3′ short overhang to allow reverse transcription, a stem region for hot start and a template-specific region for PCR amplification. As proof of principle, GAPDH, SARS-CoV-2 synthetic RNA and SARS-CoV-2 virus-positive nasopharyngeal swabs were used as templates. Additionally, these primers were used to positively confirm the N501Y mutation from nasopharyngeal swabs. Evidence is presented that the double-bubble primers offer fast, specific, robust and cost-effective improvement in RT-PCR amplification for detection of gene expression in general and for diagnostic detection and genotyping of SARS-CoV-2 in particular.

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A New Approach to Identify the Methylation Sites in the Control Region of Mitochondrial DNA.

Current Molecular Medicine ◽

10.2174/1566524020666200528154005 ◽

2020 ◽

Vol 20 ◽

Author(s):

Ashael Alfredo Pérez-Muñoz ◽

María de Lourdes Muñoz ◽

Normand García-Hernández ◽

Heriberto Santander-Lucio

Keyword(s):

Mitochondrial Dna ◽

Control Region ◽

Pcr Amplification ◽

Regulatory Elements ◽

Negative Control ◽

Human Populations ◽

Rt Pcr ◽

New Approach ◽

Cpg Dinucleotides ◽

Bisulfite Treatment

: Mitochondrial DNA (mtDNA) methylation has the potential to be used as a biomarker of human development or disease. However, mtDNA methylation procedures are costly and time-consuming. Therefore, we developed a new approach based on an RT-PCR assay for the base site identification of methylated cytosine in the control region of mtDNA in a simple, fast, specific, and low-cost strategy. Total DNA was purified, and methylation was determined by RT-PCR bisulfite sequencing. This procedure included the DNA purification, bisulfite treatment and RT-PCR amplification of the control region divided into three subregions with specific primers. Sequences obtained with and without the bisulfite treatment were compared to identify the methylated cytosine dinucleotides. Furthermore, the efficiency of C to U conversion of cytosines was assessed by including a negative control. Interestingly, mtDNA methylation was observed mainly within non-C-phosphate-G (non-CpG) dinucleotides and mostly in the regions containing regulatory elements, such as OH or CSBI, CSBII, and CSBIII. This new approach will promote the generation of new information regarding mtDNA methylation patterns in samples from patients with different pathologies or that are exposed to a toxic environment in diverse human populations.

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Seroprevalence and Real-time PCR Detection of Brucellosis in Abattoirs Animals as a Potential Route of Infection in Egypt

European Journal of Medical and Health Sciences ◽

10.24018/ejmed.2019.1.5.105 ◽

2019 ◽

Vol 1 (5) ◽

Author(s):

Amira Mohamed Zakaria ◽

Salwa F. Ahmed ◽

Mohamed S. Motawae

Keyword(s):

Real Time ◽

Rose Bengal ◽

Real Time Pcr ◽

Serological Test ◽

Pcr Amplification ◽

Cost Effective ◽

Rt Pcr ◽

Serum Samples ◽

Molecular Approaches ◽

Rose Bengal Test

Brucellosis is among the most common and economically serious zoonosis worldwide. Brucellosis in Egypt is an endemic problem among animals and humans. This work intended to evaluate the conventional serological and molecular approaches as a tool for studying the prevalence of brucellosis within abattoir’s animals in two large Egyptian provinces. Two hundred and thirty (n=230) blood and serum samples were collected from (2-3) years male calves in two Egyptian abattoirs. Rose Bengal test (RBT) and modified in-house ELISA were applied to determine the seroprevalence of Brucellosis in abattoirs animals while quantitative Taqman real-time PCRs (RT-PCR) were implemented for the characterization of Brucella species. The overall prevalence of brucellosis in the two proveinces was (53.9 %) , (75.2 %) and (79.1 %) as determined by ELISA ,RBT and RT- PCR assays respectively. Brucella DNA was successfully amplified from serum samples as well as blood. A total of n= 182 samples (79.1 %) were identified by real time PCR amplification for IS711 gene as Brucella genus, n= 118 (64.8 %) were reported as B. aborts while n= 85 (46.7 %) were reported as B. melitensis. N= 44 (24.17 %) from the collected samples comprised the two species of bacteria. This study endorses the application of rose Bengal test as a sensitive and cost effective serological test for brucellosis and real-time PCR as a distinguishing tool to detect the causative agents. Our findings indicate a significantly high prevalence of Brucella antibodies and DNA in blood and serum samples which poses a crucial threat to public health in Egypt.

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1376: Molecular Diagnostic Detection of Haploid Germ Cells in Testicular Biopsy Specimens by Online RT-PCR

The Journal of Urology ◽

10.1016/s0022-5347(18)38601-4 ◽

2004 ◽

Vol 171 (4S) ◽

pp. 362-363

Author(s):

Mark G. Schrader ◽

Markus Muller ◽

Wolfgang Schulze ◽

Steffen Weikert ◽

Kurt Miller

Keyword(s):

Germ Cells ◽

Testicular Biopsy ◽

Molecular Diagnostic ◽

Rt Pcr ◽

Biopsy Specimens ◽

Diagnostic Detection

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New Approach to Non-invasive Visualisation of Cardiac Function – Riskfree Test Can Provide Fast, Cost-effective Diagnosis

European Cardiology Review ◽

10.15420/ecr.2006.1.1t ◽

2006 ◽

Vol 2 (1) ◽

pp. 1

Author(s):

CardioMag Imaging

Keyword(s):

Cardiac Function ◽

Cost Effective ◽

New Approach ◽

Non Invasive ◽

Effective Diagnosis

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Janus – A New Approach to Air Combat Pilot Training

Transactions on Aerospace Research ◽

10.2478/tar-2019-0019 ◽

2019 ◽

Vol 2019 (4) ◽

pp. 7-22

Author(s):

Georges Bridel ◽

Zdobyslaw Goraj ◽

Lukasz Kiszkowiak ◽

Jean-Georges Brévot ◽

Jean-Pierre Devaux ◽

...

Keyword(s):

Low Cost ◽

Cost Effective ◽

High Energy ◽

Training System ◽

Embedded Sensors ◽

Pilot Training ◽

New Approach ◽

Combat Aircraft ◽

Air Combat ◽

The Cost

Abstract Advanced jet training still relies on old concepts and solutions that are no longer efficient when considering the current and forthcoming changes in air combat. The cost of those old solutions to develop and maintain combat pilot skills are important, adding even more constraints to the training limitations. The requirement of having a trainer aircraft able to perform also light combat aircraft operational mission is adding unnecessary complexity and cost without any real operational advantages to air combat mission training. Thanks to emerging technologies, the JANUS project will study the feasibility of a brand-new concept of agile manoeuvrable training aircraft and an integrated training system, able to provide a live, virtual and constructive environment. The JANUS concept is based on a lightweight, low-cost, high energy aircraft associated to a ground based Integrated Training System providing simulated and emulated signals, simulated and real opponents, combined with real-time feedback on pilot’s physiological characteristics: traditionally embedded sensors are replaced with emulated signals, simulated opponents are proposed to the pilot, enabling out of sight engagement. JANUS is also providing new cost effective and more realistic solutions for “Red air aircraft” missions, organised in so-called “Aggressor Squadrons”.

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Detection of enteroviruses in marine waters by direct RT-PCR and cell culture

Water Science & Technology ◽

10.2166/wst.1995.0635 ◽

1995 ◽

Vol 31 (5-6) ◽

pp. 323-328 ◽

Cited By ~ 8

Author(s):

K. A. Reynolds ◽

C. P. Gerba ◽

I. L. Pepper

Keyword(s):

Cell Culture ◽

Polymerase Chain Reaction Amplification ◽

Cost Effective ◽

The United States ◽

Water Runoff ◽

Rt Pcr ◽

Marine Waters ◽

Storm Water Runoff ◽

Routine Monitoring ◽

Reaction Amplification

Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptase-polymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 × 25 cycles) in 10 μL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.

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Monitoring the marine environment for small round structured viruses (SRSVS): a new approach to combating the transmission of these viruses by molluscan shellfish

Water Science & Technology ◽

10.2166/wst.1998.0498 ◽

1998 ◽

Vol 38 (12) ◽

pp. 51-56 ◽

Cited By ~ 12

Author(s):

K. Henshilwood ◽

J. Green ◽

D. N. Lees

Keyword(s):

Enteric Virus ◽

Winter Period ◽

Rt Pcr ◽

Cloning And Sequencing ◽

New Approach ◽

Human Enteric Virus ◽

Different Strains ◽

The Uk ◽

Public Health Protection

This study investigates human enteric virus contamination of a shellfish harvesting area. Samples were analysed over a 14-month period for Small Round Structured Viruses (SRSVs) using a previously developed nested RT-PCR. A clear seasonal difference was observed with the largest numbers of positive samples obtained during the winter period (October to March). This data concurs with the known winter association of gastroenteric illness due to oyster consumption in the UK and also with the majority of the outbreaks associated with shellfish harvested from this area during the study period. RT-PCR positive amplicons were further characterised by cloning and sequencing. Sequence analysis of the positive samples identified eleven SRSV strains, of both Genogroup I and Genogroup II, occurring throughout the study period. Many shellfish samples contained a mixture of strains with a few samples containing up to three different strains with both Genogroups represented. The observed common occurrence of strain mixtures may have implications for the role of shellfish as a vector for dissemination of SRSV strains. These results show that nested RT-PCR can identify SRSV contamination in shellfish harvesting areas. Virus monitoring of shellfish harvesting areas by specialist laboratories using RT-PCR is a possible approach to combating the transmission of SRSVs by molluscan shellfish and could potentially offer significantly enhanced levels of public health protection.

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Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq

Nature Communications ◽

10.1038/s41467-021-22664-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ramesh Yelagandula ◽

◽

Aleksandr Bykov ◽

Alexander Vogt ◽

Robert Heinen ◽

...

Keyword(s):

Influenza A ◽

Respiratory Infections ◽

High Sensitivity ◽

Cost Effective ◽

Massively Parallel ◽

Rt Pcr ◽

Viral Spread ◽

Saliva Analysis ◽

Double Blinded ◽

Generation Sequencing

AbstractThe COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

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