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      Amplification chemistries in clinical virology

      review-article
      , *
      Journal of Clinical Virology
      Elsevier B.V.
      PCR, LAMP, HDA, Amplification, NEAR, NASBA

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          Highlights

          • This review article describes the amplification chemistries that are commonly used in the clinical virology laboratory.

          • The article includes different techniques for target, signal, and probe amplification chemistries.

          • Major features and limitations of each chemistry are described.

          • Commercial applications of these chemistries for viral pathogens that infect humans are included.

          Abstract

          Molecular diagnostic methods have evolved and matured considerably over the last several decades and are constantly being evaluated and adopted by clinical laboratories for the identification of infectious pathogens. Advancement in other technologies such as fluorescence, electronics, instrumentation, automation, and sensors have made the overall diagnostic process more accurate, sensitive, and rapid. Nucleic acid based detection procedures, which rely on the fundamental principles of DNA replication have emerged as a popular and standard diagnostic method, and several commercial assays are currently available based on different nucleic acid amplification techniques. This review focuses on the major amplification chemistries that are used for developing commercial assays and discusses their application in the clinical virology laboratory.

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          Most cited references93

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          Point-of-care nucleic acid testing for infectious diseases.

          Nucleic acid testing for infectious diseases at the point of care is beginning to enter clinical practice in developed and developing countries; especially for applications requiring fast turnaround times, and in settings where a centralized laboratory approach faces limitations. Current systems for clinical diagnostic applications are mainly PCR-based, can only be used in hospitals, and are still relatively complex and expensive. Integrating sample preparation with nucleic acid amplification and detection in a cost-effective, robust, and user-friendly format remains challenging. This review describes recent technical advances that might be able to address these limitations, with a focus on isothermal nucleic acid amplification methods. It briefly discusses selected applications related to the diagnosis and management of tuberculosis, HIV, and perinatal and nosocomial infections. Copyright © 2011. Published by Elsevier Ltd.
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            Is Open Access

            FilmArray, an Automated Nested Multiplex PCR System for Multi-Pathogen Detection: Development and Application to Respiratory Tract Infection

            The ideal clinical diagnostic system should deliver rapid, sensitive, specific and reproducible results while minimizing the requirements for specialized laboratory facilities and skilled technicians. We describe an integrated diagnostic platform, the “FilmArray”, which fully automates the detection and identification of multiple organisms from a single sample in about one hour. An unprocessed biologic/clinical sample is subjected to nucleic acid purification, reverse transcription, a high-order nested multiplex polymerase chain reaction and amplicon melt curve analysis. Biochemical reactions are enclosed in a disposable pouch, minimizing the PCR contamination risk. FilmArray has the potential to detect greater than 100 different nucleic acid targets at one time. These features make the system well-suited for molecular detection of infectious agents. Validation of the FilmArray technology was achieved through development of a panel of assays capable of identifying 21 common viral and bacterial respiratory pathogens. Initial testing of the system using both cultured organisms and clinical nasal aspirates obtained from children demonstrated an analytical and clinical sensitivity and specificity comparable to existing diagnostic platforms. We demonstrate that automated identification of pathogens from their corresponding target amplicon(s) can be accomplished by analysis of the DNA melting curve of the amplicon.
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              Real-time PCR in the microbiology laboratory.

              I MacKay (2004)
              Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.
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                Author and article information

                Contributors
                Journal
                J Clin Virol
                J. Clin. Virol
                Journal of Clinical Virology
                Elsevier B.V.
                1386-6532
                1873-5967
                27 March 2019
                June 2019
                27 March 2019
                : 115
                : 18-31
                Affiliations
                [0005]Luminex Corporation, Austin, TX, United States
                Author notes
                [* ]Corresponding author at: 12212 Technology Blvd, Austin, TX, 78727, United States. sdas@ 123456luminexcorp.com
                Article
                S1386-6532(19)30071-X
                10.1016/j.jcv.2019.03.015
                7106405
                30953805
                9ca1420f-6110-4be7-bea6-f2ef7693e005
                © 2019 Elsevier B.V. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 14 November 2018
                : 19 February 2019
                : 25 March 2019
                Categories
                Article

                Microbiology & Virology
                pcr,lamp,hda,amplification,near,nasba
                Microbiology & Virology
                pcr, lamp, hda, amplification, near, nasba

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