Molecular Biology of PCR Testing for COVID-19 Diagnostics

2021 ◽  
Author(s):  
Vinita Chittoor-Vinod
Keyword(s):  
Molecular Biology ◽  
Respiratory Disease ◽  
Diagnostic Tests ◽  
Genetic Material ◽  
Molecular Diagnostic ◽  
Basic Principles ◽  
Asymptomatic Carriers ◽  
Diagnostic Assays ◽  
Global Pandemic ◽  
Asymptomatic Individuals

COVID-19 cases were first reported in December 2019, and since then it has spread quickly to create a global pandemic. This respiratory disease is caused by the SARS-CoV-2 virus. A major contributing factor for the fast spread of this virus is that the infectivity by the asymptomatic carriers is similar to symptomatic patients. Thus, to identify the asymptomatic individuals and to provide the essential treatment and care to COVID-19 patients, we rely heavily on diagnostic assays. Efficient, reproducible and accessible diagnostic tests are crucial in combatting a pandemic. Currently, there are few key detection tests which have been successfully employed to field-use. However, there are constant efforts to enhance their efficacy and accessibility. This chapter aims at explaining the basic principles of the current molecular diagnostic tests, which determine the presence of the virus through the detection of its genetic material. This chapter will aid the readers in understanding the basic workings of these molecular diagnostic tests.

Combating the progression of novel coronavirus SARS-CoV-2 infectious Disease: Current state and future prospects in molecular diagnostic and drug discovery

2021 ◽  
Vol 21 ◽  
Author(s):  
Arbind Kumar ◽  
Aashish Sharma ◽  
Narendra Vijay Tirpude ◽  
Sharad Thakur ◽  
Sanjay Kumar
Keyword(s):  
Serological Test ◽  
Clinical Stage ◽  
Genetic Material ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Future Prospects ◽  
Diagnostic Assays ◽  
Global Pandemic ◽  
Life Threatening ◽  
Novel Coronavirus

: A highly infectious and life-threatening virus originated from Wuhan, China in late 2019 and rapidly spread all over the world. This novel virus belongs to coronavirus family and is associated with severe acute respiratory syndrome (SARS), causing respiratory disease known as COVID-19. In March 2020, WHO has declared COVID-19 outbreak a global pandemic. Morbidity and mortality are swiftly rising day by day with the situation more severe and fatal for the co-morbid population. Many COVID-19 patients are asymptomatic, but they are silently spreading the infection. There is a need for proper screening of infected patients to prevent the epidemic transmission of disease and also for the early curative interventions to reduce the risk of developing serious complications from COVID-19 illness. To date, the diagnostic assays fall under two categories, molecular detection of viral genetic material by realtime RT-polymerase chain reaction and serological test, which rely on detecting antiviral antibodies. Unfortunately, there are no effective prophylactics and therapeutics available against COVID-19. However, few drugs are showing promising antiviral activity against it and presently are referred for the clinical trials, albeit FDA issued an Emergency Use Authorization (EUA) for emergency use of few drugs in the manifestation of SARS-CoV-2 infection. This review gives an insight into current progress, challenges and future prospects of laboratory detection methods of COVID-19 and also highlights the clinical stage of the major proposed or evidence based drugs/vaccines recommended against the novel SARS-CoV-2 pandemic virus.

scholarly journals Molecular diagnosis of B- and T-cell lymphomas: fundamental principles and clinical applications

1997 ◽  
Vol 43 (10) ◽  
pp. 1814-1823 ◽  
Author(s):  
William N Rezuke ◽  
Evelyn C Abernathy ◽  
Gregory J Tsongalis
Keyword(s):  
T Cell ◽  
Residual Disease ◽  
Molecular Diagnostic ◽  
Basic Principles ◽  
Diagnostic Assays ◽  
Lymphoid Malignancies ◽  
T Cell Lymphomas ◽  
Polymerase Chain ◽  
T Cell Clonality ◽  
Cell Clonality

Abstract Molecular diagnostic assays have become routine in the evaluation of lymphoid malignancies. Both Southern transfer and polymerase chain reaction (PCR) technologies are used to assess for B- and T-cell clonality, the presence of rearrangements involving protooncogenes such as bcl-1 and bcl-2, and the monitoring of minimal residual disease. We review the fundamentals of B- and T-cell ontogeny as well as the basic principles of the Southern transfer and PCR assays and their applications to the diagnosis of lymphoid malignancies.

Laboratory Methods for the Diagnosis of SARS-Cov-2

2022 ◽  
pp. 58-77
Author(s):  
Mohamed Echchakery ◽  
Samia Boussaa ◽  
Souad El Mouahid ◽  
Maryam Mountassir ◽  
Said El Hizazi ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Diagnostic Tests ◽  
Epidemiological Studies ◽  
Virus Antigen ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Laboratory Methods ◽  
Virus Surface ◽  
Diagnostic Assays ◽  
Human Endeavor

The coronavirus disease 2019 (COVID-19) which has become the pandemic par excellence of our time places pressure on various aspects of human endeavor and as such requires detailed study to better combat it. However, diagnostic tests were used to provide data on the incidence of COVID-19 and to assess the immune status of infected individuals. The objective of this chapter is to describe the diagnostic methods currently used to identify SARS-CoV-2 infection. Obtaining the first SARS-CoV-2 genome sequence was decisive for the development of molecular diagnostic assays that currently make it possible to diagnose and screen for the Sars-CoV-2 infection. Their uses depend on the target to be detected. Antigenic tests detect the presence of a virus antigen, which usually makes a proteinaceous part of the virus surface. The serology tests detect the presence of antibodies generated against SARS-CoV-2 and are also a relevant tool for epidemiological studies.

scholarly journals Molecular biology and respiratory disease. I-- Basic principles.

Thorax ◽  
1990 ◽  
Vol 45 (1) ◽  
pp. 52-56
Author(s):  
C A Owen ◽  
R A Stockley
Keyword(s):  
Molecular Biology ◽  
Respiratory Disease ◽  
Basic Principles

scholarly journals Comparison of Serological and Molecular Assays for Bartonella Species in Dogs with Hemangiosarcoma

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 794
Author(s):  
Erin Lashnits ◽  
Pradeep Neupane ◽  
Julie M. Bradley ◽  
Toni Richardson ◽  
Ricardo G. Maggi ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Latent Class ◽  
Fluorescent Antibody ◽  
Molecular Diagnostic ◽  
Infection Prevalence ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Tissue Biopsy ◽  
Frozen Tissue ◽  
Fresh Frozen

Currently, a gold standard diagnostic test for Bartonella infection in dogs is lacking. This represents a critical limitation for the development and evaluation of new diagnostic tests, as well as for the diagnosis of, and research on, bartonellosis in dogs. This retrospective observational study aims to compare the results of commonly performed and newly-reported Bartonella spp. diagnostic tests in banked clinical specimens from 90 dogs with hemangiosarcoma (HSA) using composite reference standard (CRS) and random effects latent class analysis (RE-LCA) techniques. Samples from each dog were tested using six serological or molecular diagnostic assays, including indirect fluorescent antibody (IFA) and Western blot (WB) for the detection of antibodies in serum, and qPCR and droplet digital PCR (ddPCR) in blood and fresh frozen tissue biopsy samples (mainly splenic HSA tumors and histopathologically normal spleen or skin/adipose tissue). Bartonella infection prevalence was estimated to be 78% based on the CRS (parallel testing with all six assays), and 64% based on the RE-LCA model. The assay with the highest diagnostic accuracy was qPCR performed on fresh frozen tissue biopsy samples (sensitivity: 94% by RE-LCA and 80% by CRS; specificity: 100%). When comparing newly-reported to traditional Bartonella diagnostic assays, ddPCR was more sensitive for the detection of Bartonella DNA than qPCR when testing blood samples (36% vs. 0%, p < 0.0001). Dogs that were positive on serological assays alone with negative molecular assays were highly unlikely (<3%) to be classified as infected by the RE-LCA model. These data indicate that Bartonella spp. DNA can be PCR amplified from fresh frozen tissues from a majority of dogs with HSA using both qPCR and ddPCR, supporting the use of these methods for future controlled studies comparing the prevalence of Bartonella spp. DNA in the tissue of dogs with HSA to that of unaffected controls.

scholarly journals Analysis of the potential impact of genomic variants in SARS-CoV-2 genomes from India on molecular diagnostic assays

2020 ◽  
Author(s):  
Abhinav Jain ◽  
Mercy Rophina ◽  
Saurabh Mahajan ◽  
Bhavya Balaji Krishnan ◽  
Manasa Sharma ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Binding Sites ◽  
Molecular Diagnostic ◽  
Emerging Pathogens ◽  
Diagnostic Assays ◽  
Genomic Variants ◽  
International Agencies ◽  
Global Pandemic ◽  
Variant Frequency ◽  
Potential Impact

ABSTRACTAn isolated epidemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus Diseases (COVID-19) originating in Wuhan, China has now rapidly emerged into a global pandemic affecting millions of people worldwide. Molecular detection of SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) forms the mainstay in screening, diagnosis and epidemiology of disease. The virus has been evolving through base substitutions. The recent availability of genomes of SARS-CoV-2 isolates from different countries including India motivated us to assess the presence and potential impact of variations in target sites for the oligonucleotide primers and probes used in molecular diagnosis. We catalogued a total of 132 primers or probes sequences from the literature and the public domain. Our analysis revealed a total of 125 unique genetic variants in 80 either primers or probes binding sites. A total of 13 unique variants had allele frequency of ≥ 1% in Indian SARS-CoV-2 genomes mapped to the primers or probes binding sites. A total of 15 primers or probes binding sites had cumulative variant frequency of ≥ 1% in the SARS-CoV-2 genomes. These included primers or probes sites which are widely used in India and across the world for molecular diagnosis as well as approved by national and international agencies. This highlights the need for sequencing genomes of emerging pathogens to make evidence based policies for development and approval of diagnostics. To the best of our knowledge, ours is the most comprehensive analysis of genomic variants in genomes of SARS-CoV-2 isolates from India and their potential impact on efficacy of molecular diagnostics.

scholarly journals Implementation of Rapid Molecular Diagnostic Tests and Antimicrobial Stewardship Involvement in Acute-Care Hospitals

2020 ◽  
Vol 41 (S1) ◽  
pp. s278-s279
Author(s):  
Maiko Kondo ◽  
Matthew Simon ◽  
Esther Babady ◽  
Angela Loo ◽  
David Calfee
Keyword(s):  
Antimicrobial Stewardship ◽  
Diagnostic Tests ◽  
New Technology ◽  
Clinical Care ◽  
Bloodstream Infections ◽  
Clinical Decision ◽  
Research Network ◽  
Molecular Diagnostic ◽  
Financial Outcomes ◽  
Stewardship Program

Background: In recent years, several rapid molecular diagnostic tests (RMDTs) for infectious diseases diagnostics, such as bloodstream infections (BSIs), have become available for clinical use. The extent to which RMDTs have been adopted and how the results of these tests have been incorporated into clinical care are currently unknown. Methods: We surveyed members of the Society for Healthcare Epidemiology of America Research Network to characterize utilization of RMDT in hospitals and antimicrobial stewardship program (ASP) involvement in result communication and interpretation. The survey was administered using Qualtrics software, and data were analyzed using Stata and Excel software. Results: Overall, 57 responses were received (response rate, 59%), and 72% were from academic hospitals; 50 hospitals (88%) used at least 1 RMDT for BSI (Fig. 1). The factors most commonly reported to have been important in the decision to adopt RMDT were improvements in antimicrobial usage (82%), clinical outcomes (74%), and laboratory efficiency (52%). Among 7 hospitals that did not use RMDT for BSI, the most common reason was cost of new technology. In 50 hospitals with RMDT for BSI, 54% provided written guidelines for optimization or de-escalation of antimicrobials based upon RMDT results. In 40 hospitals (80%), microbiology laboratories directly notified a healthcare worker of the RMDT results: 70% provided results to a physician, nurse practitioner, or physician assistant; 48% to the ASP team; and 33% to a nurse. Furthermore, 11 hospitals (22%) had neither guidelines nor ASP intervention. In addition, 24 hospitals (48%) reported performing postimplementation evaluation of RMDT impact. Reported findings included reduction in time to antibiotic de-escalation (75%), reduction in length of stay (25%), improved laboratory efficiency (20%), and reduction in mortality and overall costs (12%). Among the 47 hospitals with both RMDT and ASP, 79% reported that the ASP team routinely reviewed blood culture RMDT results, and 53.2% used clinical decision support software to do so. Finally, 53 hospitals (93%) used 1 or more RMDT for non–bloodstream infections (Fig. 1). Fewer than half of hospitals provided written guidelines to assist clinicians in interpreting these RMDT results. Conclusions: RMDTs have been widely adopted by participating hospitals and are associated with positive self-reported clinical, logistic, and financial outcomes. However, nearly 1 in 4 hospitals did not have guidelines or ASP interventions to assist clinicians with optimization of antimicrobial prescribing based on RMDT results for BSI. Also, most hospitals did not have guidelines for RMDT results for non-BSI. These findings suggest that opportunities exist to further enhance the potential benefits of RMDT.Funding: NoneDisclosures: None

scholarly journals Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

2007 ◽  
Vol 88 (2) ◽  
pp. 621-630 ◽  
Author(s):  
S. Maan ◽  
N. S. Maan ◽  
A. R. Samuel ◽  
S. Rao ◽  
H. Attoui ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Full Length ◽  
Molecular Diagnostic ◽  
Mammalian Host ◽  
Reading Frame ◽  
Diagnostic Assays ◽  
Primary Determinant ◽  
Serotype Identification

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically ‘related’. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

scholarly journals Use of Quantitative Molecular Diagnostic Assays to Investigate Fusarium Dry Rot in Potato Stocks and Soil

2005 ◽  
Vol 95 (12) ◽  
pp. 1462-1471 ◽  
Author(s):  
D. W. Cullen ◽  
I. K. Toth ◽  
Y. Pitkin ◽  
N. Boonham ◽  
K. Walsh ◽  
...  
Keyword(s):  
Control Strategies ◽  
Disease Incidence ◽  
Harvest Date ◽  
Molecular Diagnostic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potato Seed ◽  
Fusarium Spp ◽  
Dry Rot ◽  
Diagnostic Assays ◽  
Final Harvest

Specific and sensitive quantitative diagnostics, based on real-time (TaqMan) polymerase chain reaction (PCR) and PCR enzyme-linked immunosorbent assay, were developed to detect dry-rot-causing Fusarium spp. (F. avenaceum, F. coeruleum, F. culmorum, and F. sulphureum). Each assay detected Fusarium spp. on potato seed stocks with equal efficiency. Four potato stocks, sampled over two seed generations from Scottish stores, were contaminated with F. avenaceum, F. sulphureum, F. culmorum, F. coeruleum or a combination of species, and there was a general trend towards increased Fusarium spp. contamination in the second generation of seed sampled. F. sulphureum and F. coeruleum caused significantly (P < 0.05) more disease in storage than the other species when disease-free tubers of potato cvs. Spunta and Morene were inoculated at a range of inoculum concentrations (0, 104, 105, and 106 conidia/ml). Increased DNA levels were correlated with increased disease severity between 8 and 12 weeks of storage. The threshold inoculum levels resulting in significant disease development on both cultivars were estimated to be 104 conidia/ml for F. sulphureum and 105 conidia/ml for F. coeruleum. To study the effect of soil infestation and harvest date on disease incidence, seed tubers of cvs. Morene and Spunta were planted in a field plot artificially infested with the four Fusarium spp. F. culmorum and F. sulphureum were detected in soil taken from these plots at harvest, and F. sulphureum DNA levels increased significantly (P < 0.05) at the final harvest. All four Fusarium spp. were detected in progeny tubers. There was a trend toward higher levels of F. culmorum detected in progeny tubers at the earliest harvest date, and higher levels of F. sulphureum at the final harvest. The use of diagnostic assays to detect fungal storage rot pathogens and implications for disease control strategies are discussed.

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