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Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance.

ABSTRACT: A point-of-care HIV-1 genotypic resistance assay that could be performed during a clinic visit would enable care providers to make informed treatment decisions for patients starting therapy or experiencing virologic failure on therapy. The main challenge for such an assay is the genetic variability at and surrounding each drug-resistance mutation (DRM). We analyzed a database of diverse global HIV sequences and used thermodynamic simulations to design an array of surface-bound oligonucleotide probe sets with each set sharing distinct 5' and 3' flanking sequences but having different centrally located nucleotides complementary to six codons at HIV-1 DRM reverse transcriptase position 103: AAA, AAC, AAG, AAT, AGA, and AGC. We then performed in vitro experiments using 80-mer oligonucleotides and PCR-amplified DNA from clinical plasma HIV-1 samples and culture supernatants that contained subtype A, B, C, D, CRF01_AE, and CRF02_AG viruses. Multiplexed solid-phase melt curve analysis discriminated perfectly among each of the six reported reverse transcriptase position 103 codons in both 80-mers and clinical samples. The sensitivity and specificity for detecting targets that contained AAC mixed with targets that contained AAA were >98% when AAC was present at a proportion of ≥10%. Multiplexed solid-phase melt curve analysis is a promising approach for developing point-of-care assays to distinguish between different codons in genetically variable regions such as those surrounding HIV-1 DRMs.

SUBMITTER: Clutter DS

PROVIDER: S-EPMC6643098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recently, illicit drug use has become more widespread and is linked to problems with crime and public health. These drugs disrupt consciousness, affecting perceptions and feelings. Combining stimulants and depressants to suppress the effect of drugs has become the most common reason for drug overdose deaths. On-site platforms for illicit-drug detection have gained an important role in dealing, without any excess equipment, long process, and training, with drug abuse and drug trafficking. Consequently, the development of rapid, sensitive, noninvasive, and reliable multiplex drug-detecting platforms has become a major necessity. In this study, a multiplex laser-scribed graphene (LSG) sensing platform with one counter, one reference, and three working electrodes was developed for rapid and sensitive electrochemical detection of amphetamine (AMP), cocaine (COC), and benzodiazepine (BZD) simultaneously in saliva samples. The multidetection sensing system was combined with a custom-made potentiostat to achieve a complete point-of-care (POC) platform. Smartphone integration was achieved by a customized application to operate, display, and send data. To the best of our knowledge, this is the first multiplex LSG-based electrochemical platform designed for illicit-drug detection with a custom-made potentiostat device to build a complete POC platform. Each working electrode was optimized with standard solutions of AMP, COC, and BZD in the concentration range of 1.0 pg/mL-500 ng/mL. The detection limit of each illicit drug was calculated as 4.3 ng/mL for AMP, 9.7 ng/mL for BZD, and 9.0 ng/mL for COC. Healthy and MET (methamphetamine) patient saliva samples were used for the clinical study. The multiplex LSG sensor was able to detect target analytes in real saliva samples successfully. This multiplex detection device serves the role of a practical and affordable alternative to conventional drug-detection methods by combining multiple drug detections in one portable platform.

Project description:BackgroundMethylation of DNA is a common mechanism for silencing genes, and aberrant methylation is increasingly being implicated in many diseases such as cancer. There is a need for robust, inexpensive methods to quantitate methylation across a region containing a number of CpGs. We describe and validate a rapid, in-tube method to quantitate DNA methylation using the melt data obtained following amplification of bisulfite modified DNA in a real-time thermocycler.MethodsWe first describe a mathematical method to normalise the raw fluorescence data generated by heating the amplified bisulfite modified DNA. From this normalised data the temperatures at which melting begins and finishes can be calculated, which reflect the less and more methylated template molecules present respectively. Also the T50, the temperature at which half the amplicons are melted, which represents the summative methylation of all the CpGs in the template mixture, can be calculated. These parameters describe the methylation characteristics of the region amplified in the original sample.ResultsFor validation we used synthesized oligonucleotides and DNA from fresh cells and formalin fixed paraffin embedded tissue, each with known methylation. Using our quantitation we could distinguish between unmethylated, partially methylated and fully methylated oligonucleotides mixed in varying ratios. There was a linear relationship between T50 and the dilution of methylated into unmethylated DNA. We could quantitate the change in methylation over time in cell lines treated with the demethylating drug 5-aza-2'-deoxycytidine, and the differences in methylation associated with complete, clonal or no loss of MGMT expression in formalin fixed paraffin embedded tissues.ConclusionWe have validated a rapid, simple in-tube method to quantify methylation which is robust and reproducible, utilizes easily designed primers and does not need proprietary algorithms or software. The technique does not depend on any operator manipulation or interpretation of the melt curves, and is suitable for use in any laboratory with a real-time thermocycler. The parameters derived provide an objective description and quantitation of the methylation in a specimen, and can be used to for statistical comparisons of methylation between specimens.

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Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance.

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