scholarly journals Using gel-electrophoresis and traditional PCR in RT-PCR assays for the detection of SARS-CoV-2 in resource limited areas

2020 ◽  
Author(s):  
Franz-Josef Wischmann ◽  
Frank Hünger ◽  
Uwe Cassens ◽  
Wolfgang Göhde
Keyword(s):  
Gel Electrophoresis ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Test Analysis ◽  
Resource Limited ◽  
Freeze Dried ◽  
Test Kit ◽  
Nucleic Acid Amplification Technology ◽  
Laboratory Technology

Background. qRT-PCR investigations on patient specimen seem to be the only probable laboratory technology to validate the current individual SARS-CoV-2 status correctly. This kind of nucleic acid amplification technology is available in most countries, but missing in resource limited areas with poor laboratory infrastructure. The most feasible way to build up PCR test capacities for SARS-CoV-2 in these areas is the use of conventional PCR. PCR-thermocycler and gel-electrophoresis systems could be shipped easily by parcel service to the whole world, because they are almost maintenance free and need no engineers for their installation and service. Methods. In order to reduce the complexity of sensitive reagent kits we have developed a freeze dried test kit for the traditional RT-PCR for the detection of SARS-CoV-2 in single ready to use tubes. Transport and storage of the reagents are now free of any cooling chain.Results. We have analysed 244 patient samples and compared the results to the SEEGENE Allplex™ 2019-nCoV assay. The sensitivity and specificity were 95.5% and 98.5%, respectively. Next to these performance parameters we could implement a workflow for processing 96 patient samples in parallel, handled by only 1 laboratory technician in an 8 hours working day.Conclusion. The use of conventional PCR in combination with gel-electrophoresis for test analysis is a feasible approach to make SARS-CoV-2 diagnostic available in the whole world. This is needed urgently, because SARS-CoV-2 is a question of global health and not only of the well-equipped countries.

scholarly journals Pulse-Controlled Amplification–A new powerful tool for on-site diagnostics under resource limited conditions

2021 ◽  
Vol 15 (1) ◽  
pp. e0009114
Author(s):  
Katharina Müller ◽  
Sarah Daßen ◽  
Scott Holowachuk ◽  
Katrin Zwirglmaier ◽  
Joachim Stehr ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Molecular Diagnostics ◽  
Nucleic Acid Amplification ◽  
Neglected Diseases ◽  
Target Sequence ◽  
Infectious Agents ◽  
Fast Heating ◽  
Resource Limited ◽  
Nucleic Acid Amplification Technology ◽  
Rapid Tests

Background Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity. Methods/Principle findings Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis. Conclusions/Significance PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests.

scholarly journals A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits

2020 ◽  
Vol 295 (46) ◽  
pp. 15438-15453 ◽  
Author(s):  
Samantha J. Mascuch ◽  
Sara Fakhretaha-Aval ◽  
Jessica C. Bowman ◽  
Minh Thu H. Ma ◽  
Gwendell Thomas ◽  
...  
Keyword(s):  
Support Group ◽  
Diagnostic Testing ◽  
The Novel ◽  
Rt Pcr ◽  
Research Teams ◽  
Environmental Testing ◽  
Resource Limited ◽  
Test Kit ◽  
Institute Of Technology ◽  
Novel Coronavirus

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

scholarly journals A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care

2020 ◽  
Author(s):  
Diem Hong Tran ◽  
Hoang Quoc Cuong ◽  
Hau Thi Tran ◽  
Uyen Phuong Le ◽  
Hoang Dang Khoa Do ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Point Of Care ◽  
Nucleic Acid Amplification ◽  
Gold Standard Method ◽  
Synthetic Dna ◽  
Isothermal Nucleic Acid Amplification ◽  
Freeze Dried ◽  
Test Kit

ABSTRACTThe COVID-19, caused by the novel coronavirus SARS-CoV-2, has broken out of control all over the globe and put the majority of the world under lockdown. There have been no specific antiviral medications for SARS-CoV-2 while vaccines are still under development. Thus, rapid diagnosis and necessary public health measures are currently key parts to contain the pandemic. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection. However, this method is not suitable for point-of-care (POC) diagnosis because of the timeconsuming procedure, the requirements of biosafety conditions and expensive equipment. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were developed and compared. The three methods exhibited similar performance with the limit of detection (LOD) as low as just 1 copy per reaction when evaluated on the synthetic DNA fragments. The results can be read with naked eyes within 30 minutes without crossreactivity to closely related coronaviruses. When tested with SARS-CoV-2 extracted genomic-RNA, LAMP outperformed both CPA and PSR assays. Moreover, the direct detection of SARS-CoV-2 in simulated patient samples (oropharyngeal and nasopharyngeal swabs) by colorimetric iNAATs was also successful. Further preparation of the lyophilized reagents for LAMP reactions revealed that the freeze-dried, ready-to-use kit maintained the sensitivity and LOD value of the liquid assays. These results strongly indicate that the colorimetric lyophilized LAMP test kit developed herein is highly suitable for detecting SARS-CoV-2 at POC.

scholarly journals Quantitative real-time RT-PCR – a perspective

2005 ◽  
Vol 34 (3) ◽  
pp. 597-601 ◽  
Author(s):  
S A Bustin ◽  
V Benes ◽  
T Nolan ◽  
M W Pfaffl
Keyword(s):  
Real Time ◽  
Gel Electrophoresis ◽  
Southern Blotting ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Fluorescent Reporter ◽  
Specificity And Sensitivity ◽  
Homogeneous Assay ◽  
Polymerase Chain

The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.

scholarly journals Review of Current COVID-19 Diagnostics and Opportunities for Further Development

2021 ◽  
Vol 8 ◽  
Author(s):  
Yan Mardian ◽  
Herman Kosasih ◽  
Muhammad Karyana ◽  
Aaron Neal ◽  
Chuen-Yen Lau
Keyword(s):  
Critical Role ◽  
False Negative ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Contact Tracing ◽  
Diagnostic Tools ◽  
Rt Pcr ◽  
Resource Limited Settings ◽  
Technical Errors ◽  
Resource Limited

Diagnostic testing plays a critical role in addressing the coronavirus disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Rapid and accurate diagnostic tests are imperative for identifying and managing infected individuals, contact tracing, epidemiologic characterization, and public health decision making. Laboratory testing may be performed based on symptomatic presentation or for screening of asymptomatic people. Confirmation of SARS-CoV-2 infection is typically by nucleic acid amplification tests (NAAT), which requires specialized equipment and training and may be particularly challenging in resource-limited settings. NAAT may give false-negative results due to timing of sample collection relative to infection, improper sampling of respiratory specimens, inadequate preservation of samples, and technical limitations; false-positives may occur due to technical errors, particularly contamination during the manual real-time polymerase chain reaction (RT-PCR) process. Thus, clinical presentation, contact history and contemporary phyloepidemiology must be considered when interpreting results. Several sample-to-answer platforms, including high-throughput systems and Point of Care (PoC) assays, have been developed to increase testing capacity and decrease technical errors. Alternatives to RT-PCR assay, such as other RNA detection methods and antigen tests may be appropriate for certain situations, such as resource-limited settings. While sequencing is important to monitor on-going evolution of the SARS-CoV-2 genome, antibody assays are useful for epidemiologic purposes. The ever-expanding assortment of tests, with varying clinical utility, performance requirements, and limitations, merits comparative evaluation. We herein provide a comprehensive review of currently available COVID-19 diagnostics, exploring their pros and cons as well as appropriate indications. Strategies to further optimize safety, speed, and ease of SARS-CoV-2 testing without compromising accuracy are suggested. Access to scalable diagnostic tools and continued technologic advances, including machine learning and smartphone integration, will facilitate control of the current pandemic as well as preparedness for the next one.

scholarly journals Analysis of Correlation between the Seven Important Helicobacter pylori (H. pylori) Virulence Factors and Drug Resistance in Patients with Gastritis

2020 ◽  
Vol 2020 ◽  
pp. 1-7 ◽  
Author(s):  
Sinem Oktem-Okullu ◽  
Zehra Cekic-Kipritci ◽  
Elif Kilic ◽  
Nogayhan Seymen ◽  
Nesteren Mansur-Ozen ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Virulence Factors ◽  
Virulence Genes ◽  
Life Science ◽  
Fluoroquinolone Resistance ◽  
Rt Pcr ◽  
Conventional Pcr ◽  
Test Kit ◽  
H Pylori ◽  
The Relationship

The aim of this study is to evaluate the association between seven important H. pylori virulence factors and antibiotic resistance in patients with gastritis. H. pylori strains isolated from 33 patients with gastritis were examined. Antimicrobial susceptibilities were tested by GenoType® HelicoDR (Hain Life Science, Germany) test kit and RT-PCR. The virulence-factors were determined using conventional PCR. 39% of patients were resistant for clarithromycin and 27% of patients were resistant for fluoroquinolone. 15% of patients were resistant to both clarithromycin and fluoroquinolone. The H. pylori vacA m1/s2 genotype was the most frequent allelic combination. Patients were possessed the vacA s1, m1 (6.1%); s1, m2 (6.1%); s2, m1 (15.1%); and s2, m2 (3.0%) genotypes. 94% of patients with gastritis were positive for H. pylori napA gene. Also, there were no dupA gene-positive gastritis patients. There was no significant correlation between the vacA, cagA, oipA, hpaA, babA, napA, dupA, ureA, ureB virulence genes, clarithromycin, and fluoroquinolone resistance. Herein, we report that the relationship between the H. pylori napA gene and gastritis. Although we found a correlation between H. pylori virulence factor and clinical outcome, there is a need for further studies to enlighten the relation between H. pylori virulence genes and antibiotic resistance.

scholarly journals Combined SARS-CoV-2 nucleic acid amplification testing and respiratory virus panel RT-PCR on the Hologic Panther Fusion system

2021 ◽  
Vol 138 ◽  
pp. 104792
Author(s):  
Bryan A. Stevens ◽  
Catherine A. Hogan ◽  
Kenji O. Mfuh ◽  
Ghazala Khan ◽  
Malaya K. Sahoo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Respiratory Virus ◽  
Nucleic Acid Amplification ◽  
Fusion System ◽  
Rt Pcr ◽  
Nucleic Acid Amplification Testing

scholarly journals Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real-time RT-PCR Assay Authorized for Emergency Use in Low-Prevalence Populations for High-Throughput Testing

2020 ◽  
Vol 7 (11) ◽  
Author(s):  
Gwynngelle A Borillo ◽  
Ron M Kagan ◽  
Russell E Baumann ◽  
Boris M Fainstein ◽  
Lamela Umaru ◽  
...  
Keyword(s):  
Real Time ◽  
False Negative ◽  
False Negative Rate ◽  
Nucleic Acid Amplification ◽  
Rt Pcr ◽  
Pooled Testing ◽  
Negative Results ◽  
Single Positive ◽  
Upper Respiratory ◽  
Respiratory Specimens

Abstract Background Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real-time reverse transcription polymerase chain reaction (RT-PCR) test authorized by the US Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens. Methods Positive specimens were selected from 3 prevalence groups, 1%–3%, >3%–6%, and >6%–10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with 3 negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3091 positive specimens. Results PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r = 0.96–0.99; slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90, respectively, for the N1 and N3 targets. The median Cts for 3091 positive specimens were 25.9 (N1) and 24.7 (N3). The percentage of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). Conclusions Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement, which demonstrates the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2 with a low risk of false-negative results.

scholarly journals Avian influenza and Newcastle disease virusindead chickens in markets in Dhaka, Bangladesh in 2011-2012

2017 ◽  
Vol 33 (1) ◽  
pp. 8-15
Author(s):  
LR Barman ◽  
RD Sarker ◽  
BC Das ◽  
EH Chowdhury ◽  
PM Das ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Newcastle Disease ◽  
Rapid Test ◽  
Type A ◽  
Antigen Test ◽  
Rt Pcr ◽  
Test Kit ◽  
Live Bird

A virological survey for avian influenza (AI) and Newcastle disease (ND) was conducted in two selected live bird markets (LBMs), namely Kaptan Bazar and Karwan Bazar in Dhaka city, Bangladesh from August 2011 to July 2012. A total of 513 dead chickens were collected. An immune-chromatographic rapid antigen test for Type A influenza virus and both conventional and real time RT-PCR were used for the detection and characterization of AI and ND viruses. All carcasses were first screened by the rapid antigen test kit and 93 were positive for Type A influenza virus. RT-PCR on a representative number of rapid antigen test positive samples (n = 24) confirmed the presence of Type A influenza virus and mostly H5 influenza virus (22 out of 24 tested samples). Influenza rapid test negative samples (n = 420) were subjected to routine necropsy. Heat stress, suffocation and physical injury were the most common cause of mortality (163 cases), followed by ND, suspected to be the cause of 85 deaths. On molecular investigation of these 85 samples, the presence of ND virus was confirmed in 59 and AI virus in 6; 15 were negative for both ND and AI viruses and 5 were unsuitable for investigation. Among the 59 ND confirmed cases 18 also contained AI virus. In summary, out of 513 carcasses 117 (22.81%) contained AI virus and 59 (11.50%) contained ND virus. Eighteen (3.51%) carcasses contained both AI and ND viruses. The findings suggest that both AI and ND should be considered as major threats to the poultry industry.Bangl. vet. 2016. Vol. 33, No. 1, 8-15

scholarly journals Electropherotypes and G-Types of Group A Rotaviruses Detected in Children with Diarrhea in Lagos, Nigeria

ISRN Virology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Christianah Idowu Ayolabi ◽  
David Ajiboye Ojo ◽  
George Enyimah Armah
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Migration Pattern ◽  
Genomic Diversity ◽  
Rt Pcr ◽  
Group A Rotaviruses ◽  
Group A ◽  
Stool Samples ◽  
Health Care Centers ◽  
Virus Isolates

Approximately over 500,000 children die annually due to severe dehydrating diarrhea caused by rotaviruses. This work investigated rotavirus infection among children less than 5 years with diarrhea in Lagos and determined the circulating electropherotypes and genotypes of the virus isolates. Three hundred and two (n=302) stool samples from children below 60 months were collected from different hospitals and health care centers in Lagos and subjected to enzyme immunoassay (EIA) to determine the presence of Group A rotavirus, RT-PCR to determine the G-types, and polyacrylamide gel electrophoresis (PAGE) to determine the electropherotypes. The results show that 60.3% of the samples showed distinct rotavirus RNA migration pattern, having long electropherotypes (55.3%) of seven variations dominating over the short electropherotypes (44.5%). Six different G-types were detected (G1, G2, G3, G4, G9, and G12). Serotypes G1 and G12 showed long electropherotypic pattern while G2, G3, and G9 exhibited either short or long electropherotype. All G4 detected show short electropherotypic pattern. In conclusion, information on the genomic diversity and RNA electropherotypes of rotaviruses detected in children with diarrhea in Lagos is reported in this study.

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