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Appraisal of a Simple and Effective RT-qPCR Assay for Evaluating the Reverse Transcriptase Activity in Blood Samples from HIV-1 Patients.

ABSTRACT: Testing HIV-1 RNA in plasma by PCR is universally accepted as the ultimate standard to confirm diagnosis of HIV-1 infection and to monitor viral load in patients under treatment. However, in some cases, this assay could either underestimate or overestimate the replication capacity of a circulating or latent virus. In the present study, we performed the assessment of evaluating the HIV-1 reverse transcriptase (RT) activity by means of a new assay for the functional screening of the status of HIV-1 patients. To this purpose, we utilized, for the first time on blood samples, an adapted version of a real-time RT quantitative PCR assay, utilized to evaluate the HIV-1-RT inhibitory activity of compounds. The study analyzed blood samples from 28 HIV-1-infected patients, exhibiting a wide range of viremia and immunological values. Results demonstrated that plasma HIV-1 RT levels, expressed as cycle threshold values obtained with the assay under appraisal, were inversely and highly significantly correlated with the plasma HIV-1-RNA levels of the patients. Thus, an HIV-1 RT quantitative PCR assay was created which we describe in this study, and it may be considered as a promising basis for an additional tool capable of furnishing information on the functional virological status of HIV-1-infected patients.

SUBMITTER: Macchi B

PROVIDER: S-EPMC7763350 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Appraisal of a Simple and Effective RT-qPCR Assay for Evaluating the Reverse Transcriptase Activity in Blood Samples from HIV-1 Patients.

Macchi Beatrice B Frezza Caterina C Marino-Merlo Francesca F Minutolo Antonella A Stefanizzi Valeria V Balestrieri Emanuela E Cerva Carlotta C Sarmati Loredana L Andreoni Massimo M Grelli Sandro S Mastino Antonio A

Pathogens (Basel, Switzerland) 20201213 12

Testing HIV-1 RNA in plasma by PCR is universally accepted as the ultimate standard to confirm diagnosis of HIV-1 infection and to monitor viral load in patients under treatment. However, in some cases, this assay could either underestimate or overestimate the replication capacity of a circulating or latent virus. In the present study, we performed the assessment of evaluating the HIV-1 reverse transcriptase (RT) activity by means of a new assay for the functional screening of the status of HIV- ...[more]

PMID: 33322208

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Project description:Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions-one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants and not with other major known variants. The assay's specificity was tested against a panel of respiratory pathogens (n = 16), showing high specificity toward SC-2 RNA. The assay's sensitivity was assessed using both in vitro transcribed RNA and clinical samples and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole-genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide-circulating SC-2 variants. IMPORTANCE This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic. The method can be easily scaled up and be used in high-throughput laboratories, as well as small ones. In addition to immediate help in diagnostic efforts, this method may also help in epidemiological studies focused on the spread of emerging SC-2 lineages.

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Dataset Information

Appraisal of a Simple and Effective RT-qPCR Assay for Evaluating the Reverse Transcriptase Activity in Blood Samples from HIV-1 Patients.

Publications

Appraisal of a Simple and Effective RT-qPCR Assay for Evaluating the Reverse Transcriptase Activity in Blood Samples from HIV-1 Patients.

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