Amplification chemistries in clinical virology

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. Polymerase chain reaction (PCR)The development of PCR by Kary Mullis in the 1980s revolutionized the detection of microbial pathogens in clinical specimens. PCR is a highly sensitive method that involves cyclical heating and cooling of the reaction mixture to facilitate amplification of the target DNA [3]. The method consists of three basic steps (denaturation, annealing, and extension) and the reaction mixture requires four primary components (template DNA, primers, nucleotides, and a DNA polymerase) (Fig. 1a). Double-stranded target DNA (dsDNA) is thermally denatured to form a single-stranded DNA template (ssDNA). Oligonucleotide primers anneal to complementary target sequences on the nucleic acid template and are extended by the DNA polymerase, thus creating the resultant PCR product (amplicon). The reaction occurs in a programmable thermal https://doi.

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“…Most methods are automated and provide quantitative and accurate results in a short period of time. They also eliminate the need for specialized training and reduce the risk of contamination and human error (20). These techniques can produce cost-effective, reproducible tests with high sensitivity and specificity that provide reliable diagnoses (21).…”

“…The method is fast, sensitive, does not require strong energy sources, and is easy to implement in service locations or in situations with limited resources. However, the method has some disadvantages, such as the challenge of designing compatible primer pairs, the potential to generate non-specific amplified products (20,66). Target isothermal amplification technologies include nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), loopmediated isothermal amplification (LAMP), recombinases aided amplification (RAA), and recombinase polymerase amplification (RPA), which are discussed below (67).…”

“…It is a transcription-sensitive system, ideal for specific replication of nucleic acids in vitro (69), which uses two specific oligonucleotide primers and three avian myeloblastosis virus (AMV) reverse transcriptase enzymes (70). RNAase H, RNA polymerase, and T7, together with the primers, amplify the RNA targets at 41 • C (71), producing at the end of the reaction the terminal product, ssRNA, detected by methods, such as electrochemiluminescence, gel electrophoresis, sphere-based enzymatic detection, and enzyme-linked gel assay (20).…”

“…It presents fast kinetics, producing up to 1,000 copies of target RNA per reaction. The obtained amplicons can be detected by gel electrophoresis or oligonucleotide probes (20,72). Like NASBA, it needs the reaction temperature needs to be carefully controlled to denature the secondary structures.…”

Analytical Evaluation of Visby Medical RT-PCR Portable Device for Rapid Detection of SARS-CoV-2

“…An alternative to the detection of antigen is the amplification of nucleic acid by reverse transcription in combination with isothermal methods such as: loop-mediated isothermal amplification (RT-LAMP) [ 5 , 6 , 7 ], recombinase polymerase amplification (RT-RPA) [ 8 , 9 , 10 ], and nicking-endonuclease amplification reactions (RT-NEAR) [ 11 , 12 , 13 , 14 ]; which do not need a thermal cycler, potentially enabling their use at POC facilities. The reference standard of RT-PCR also detects viral nucleic acid, and some devices capable of RT-PCR have been developed that are suitable for POC use, such as the DNAnudge Covid-19 test.…”