Applications of Real-time Reverse Transcription Polymerase Chain Reaction in Clinical Virology Laboratories for the Diagnosis of Human Diseases

Manojkumar, R.; Mrudula, V.

doi:10.3844/ajidsp.2006.204.209

Cited by 4 publications

(4 citation statements)

References 51 publications

(57 reference statements)

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“…Indeed, the speed and accuracy of diagnosis should be balanced against the test cost and its availability, as well as its adaptation to international standardized guidelines with the intention of improving quality assurance of these advanced laboratory tests. 13 In this study, specificity, precision, reportable range detection and analytical sensitivity of a RT-qPCR directed to the NS5 region from yellow fever virus were evaluated, according to previous described parameters required to validate bioanalytical methods in Brazil.…”

Section: Introductionmentioning

confidence: 99%

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

Fernandes-Monteiro

Trindade

Yamamura

et al. 2015

Human Vaccines & Immunotherapeutics

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Abbreviation: PCR, polymerase chain reaction; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; YFV, yellow fever virus; NS5, protein of the viral polyprotein, it is the largest and the most highly conserved among the flaviviral proteins; 17DD, strain used to yellow fever vaccine; YF, yellow fever; RNA, ribonucleic acid; C, capsid protein; prM/M, membrane protein; E, envelope protein; NS, nonstructural protein; ELISA, enzyme-linked immunosorbent assay; DNA, deoxyribonucleic acid; DL, detection limit; QL, quantification limit; ANVISA, Brazilian Health Surveillance Agency; CV, coefficient of variation; RNAse P, human constitutive gene; Ct, cycle threshold; EXO IPC, exogenous internal positive control; PFU, plaque former unit; FDA, food and drug administration agency; JEV, japanese encephalitis virus; DENV, dengue virus; MuV, mumps virus; MV, measles virus; MOI, multiplicity of infection; WNV, West Nile VirusThe development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

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Section: Introductionmentioning

confidence: 99%

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

Fernandes-Monteiro

Trindade

Yamamura

et al. 2015

Human Vaccines & Immunotherapeutics

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“…Amplicon detection is commonly based on enzyme‐linked gel assays, bead‐based enzymatic detection, electrochemiluminescence, molecular beacon technology, or fluorescent correlation spectroscopy (Sergentet and others ; Fakruddin and others ). Although not yet proven in food, Manojkuma and Mrudula () note that in clinical use, NASBA has a higher analytical sensitivity than RT‐PCR for pathogen detection. Although not covered here in detail, there are many other isothermal methods such as strand displacement amplification, invader assays, split DNAZyme amplification, and hybridization chain reactions (reviewed by Yan and others ).…”

Section: Pathogen Detection Schemesmentioning

confidence: 99%

Emerging Biorecognition and Transduction Schemes for Rapid Detection of Pathogenic Bacteria in Food

Vanegas

Gomes

Cavallaro

et al. 2017

Comp Rev Food Sci Food Safe

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The presence of unsafe levels of microorganisms in food constitutes a growing economic and public health problem that necessitates new technology for their rapid detection along the food continuum from production to consumption. While traditional techniques are reliable, there is a need for more sensitive, selective, rapid, and costeffective approaches for food safety evaluation. Methods such as microbiological counts are sufficiently accurate and inexpensive, and are capable of determining presence and viability for most pathogens. However, these techniques are time consuming, involve destructive sampling, and require trained personnel and biosafety-certified facilities for analysis. Molecular techniques such as the polymerase chain reaction have greatly improved analytical capability over the last decade, achieving shorter analysis time with quantitative data and strain specificity, and in some cases the ability to discriminate cell viability. The emerging field of nanosensors/biosensors has demonstrated a variety of devices that hold promise to bridge the gap between traditional plate counting and molecular techniques. Many nanosensors/biosensors are rapid, portable, accurate devices that can be used as an additional screening tool for identifying unsafe levels of microorganisms in food products with no need for pre-enrichment. In this review, we provide a brief overview of available biorecognitiontransduction techniques for detecting bacteria in food. We then discuss the advantages and disadvantages of each technique, and describe some recent biosensor or nanosensor technologies that are under development. We conclude by summarizing the opportunities and challenges in the field of pathogen monitoring in food systems and we focus the discussion on the strengths/weaknesses of the most popular biorecognition agents and transducer nanomaterials for biosensing.

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“…[ 15 ] In clinical use and pathogen detection, NASBA pose theoretically higher analytical sensitivity than reverse transcription-polymerase chain reaction RT-PCR making it an established diagnostic tool. [ 16 ] It has potential for detection and differentiation of viable cells through specific and sensitive amplification of messenger RNA, even against the background of genomic DNA. [ 17 ]…”

Section: Nucleic Acid Sequence Based Amplificationmentioning

confidence: 99%

Nucleic acid amplification: Alternative methods of polymerase chain reaction

et al. 2013

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Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

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Applications of Real-time Reverse Transcription Polymerase Chain Reaction in Clinical Virology Laboratories for the Diagnosis of Human Diseases

Cited by 4 publications

References 51 publications

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters

Emerging Biorecognition and Transduction Schemes for Rapid Detection of Pathogenic Bacteria in Food

Nucleic acid amplification: Alternative methods of polymerase chain reaction

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