Transcription mediated amplification assay, fundamentals of the technique.
Transcription mediated amplification assay (TMA) is a molecular biology methodology based on the detection of nucleic acids. Automated technique that allows solving large workloads in a short time.
In a first reaction (figure 1), the viral RNA comes into contact with a reverse transcriptase and a cDNA chain is formed. This enzyme is followed by a special RNA polymerase that will create multiple RNA amplicons through the DNA strand, which it will use as a template. Each RNA amplicon will perform the process cyclically, exponentially generating a sufficient amount of genetic load to be detected.
Analytical concordance of results of High-Throughput Transcription-mediated amplification (TMA) versus RT-PCR
Trémeaux et al. make a comparison between three different trials, pitting them one against one. In a first comparison between Aptima ™ (TMA) and Panther Fusion ™ (RT-PCR) assays after performing a contingency table and analyzing the discrepancies (table 1), they obtain a Cohen's kappa coefficient κ = 0.979,a positive percent agreement = 97.3%, a negative percent agreement = 100%, and an overall percent agreement = 99.0%. When faced with the results obtained by Aptima ™ assays versus MagNA / LC480 (RT-PCR) assays, they obtained the following concordance data, had a Cohen's coefficient κ = 0.945 a positive percent agreement = 98.6% a negative percent agreement = 96.9% and an overall percent agreement = 97.5% .Their invalid rated with the Aptima ™ assay was 0.5% (1/200).
Gorzalski et al present a comparison between two different trials. In a first comparison between Aptima ™ (TMA) versus Multiplex assay (RT-PCR) ™ assays after performing a contingency table and analyzing the discrepancies obtained (table1) a Cohen’s kappa coefficient κ = 0.98, a positive percent agreement = 98.07%, a negative percent agreement = 100%, and an overall percent agreement = 99.1% .Their invalid rated with the Aptima ™ assay was 3.44% (4/116).
This work also compares the limit of detection (table2), obtaining favorable results to Aptima™ (TMA). It is capable of detecting small amounts of viral RNA copies in the sample. In front of a concentration of 5.5 × 10e3 genomic RNA copies/m, TMA rectivity has a value of 5/5 (100%) versus 0/5 (0%) PCR reactivity. The minimum number of copies/ml that is capable of detecting the technique Aptima™ (TMA) in this work is 5.5 × 10e2 copies/mL.(Table X)
Smith et al. conducted a study comparing three techniques. Two RT-PCR assays: BioFire Defense COVID-19 test (BioFire) and the Hologic Panther Fusion SARS-CoV-2 assay (Fusion) and a TMA Aptima ™ assay. In this case, positive consensus results were obtained (when a positive result coincided in at least two of the three techniques) and a negative consensus result (when a negative result coincided in at least two of the three techniques).Then they faced the results obtained by each trial against the consensus results to obtain the concordance data.
In a first comparison (table 1) with Panther Fusion ™ (RT-PCR) and the consensus results obtain a Cohen's kappa coefficient κ = 0.987, a positive percent agreement = 98.7%, a negative percent agreement = 100%, and an overall percent agreement = 96.64% . When they compared the results obtained by Aptima ™ assays versus the consensus results, they obtained the following concordance data, had a Cohen's coefficient κ = 0.947 a positive percent agreement = 984.7% a negative percent agreement = 100% and an overall percent agreement = 97.3%. Finally, the BioFire trial versus the consensus results obtained the following concordance results, Cohen's coefficient κ = 0.987, a positive percent agreement = 98.7%, a negative percent agreement = 100% and an overall percent agreement = 99.3%.
The limit of detection of the three techniques reflects limit of detection values of 62.5 copies / mL (Fusion), 62.5 copies / mL (Aptima) and a lower limit of detection in Biofire with 125 copies / mL.(table 2).
Pham, J et al. maked a comparison between two different trials, TMA versus Panther Fusion SARS-CoV-2 RT-PCR assay and get a Cohen's kappa coefficient κ = 0.986, a positive percent agreement = 100 %, a negative percent agreement = 98.7%, and an overall percent agreement = 99.3%.
Cordes, A.K et al. comparison three techniques versus consensus results and we obtained Cohen's kappa coefficient κ = 0.986, a positive percent agreement = 100 %, a negative percent agreement = 98.7%, and an overall percent agreement = 99.3%. for Aptima SARS-CoV-2 TMA and LDT E-gene RT-PCR versus the consensus results. In the study Genesig COVID-19 RT-PCR versus the consensus results obtained Cohen's kappa coefficient κ = 0.900, a positive percent agreement = 87,5 %, a negative percent agreement = 100%, and an overall percent agreement = 95.54%.
Analytical concordance of results of High-Throughput Transcription-mediated amplification (TMA) versus Fluorescent immunoassay (FIA) for antigen detection.
Beck, E. T et al. They compare the results obtained between TMA, versus the results obtained by FIA, a test that will detect the presence of SARS-COV-2 antigen. The overall positive percent agreement (PPA), negative percent agreement (NPA), and total agreement (TA) of the SOFIA test compared to the APTIMA TMA test were 77.0% (4 7/61) 99.6% (284/285), and 95.7% (331/ 346), respectively (Table 1). In this article. The patients who participate are separated into two groups. PPA of the SOFIA test with the APTIMA TMA test was 82.0% (41/50) for patients evaluated less than 5 days from the onset of symptoms and 54.5% (6/11) for patients that exceeded 5 days from the onset of symptoms. There was also a difference when the percentage of negative agreement (NPA) and the total agreement (TA) between the two groups were compared. The results obtained were 100% versus 97.3% (NPA) and 97% versus 87.5% (TA) respectively.
Use of High-Throughput Transcription-mediated amplification (TMA) within the battery of SARS-COV-2 detection techniques in the same patient for the study of reinfections.
Tillett, R. L et al. show the sequence of tests carried out on a patient who presented symptoms compatible with SARS-COV 2 infection for the study of reinfection. The data obtained in the different tests were: positive RT-PCR (April 18), negative TMA (May 9), negative RT-PCR (May 26), positive RT-PCR (June 5) and positive IgG and IgM serology ( June 6). Through genomic sequencing of the virus, they conclude that the patient has undergone a reinfection.
Harrington, D et al. present a reinfection by Variant VOC-202012/01 that occurred in a patient from the United Kingdom. In the study they present several positive molecular diagnostic tests (RT-PCR and TMA) in different periods of time, confirming by sequencing that it is a reinfection.