One-Tube Real-Time Isothermal Amplification Assay To Identify and Distinguish Human Immunodeficiency Virus Type 1 Subtypes A, B, and C and Circulating Recombinant Forms AE and AG

Abstract

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10 3 copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.Human immunodeficiency virus type 1 (HIV-1) can be differentiated into several genetic groups and subgroups, or subtypes. The viral isolates that belong to the genetic group M are responsible for more than 99% of all infections worldwide. This group can be divided into subtypes A through H, to which subtype K was recently added (32). As for subtype J, only two strains are known, whereas subtype I appears to be a mosaic of various subtypes. Besides the group M isolates, the groups N and O are also recognized (9, 34). Isolates that belong to these two groups are mainly found in patients in Cameroon, whereas group M isolates can be found worldwide, with distinct subtypes dominant in specific geographical areas. Epidemiological data is continuously being collected ...

A novel TaqMan real-time PCR assay to estimate ex vivo human immunodeficiency virus type 1 fitness in the era of multi-target (pol and env) antiretroviral therapy

“…Lu & Kuritzkes (2001) developed a recombinant marker virus assay to perform growth competition experiments between two RT recombinant viruses which were quantified using real-time PCR for the corresponding marker. Several studies have described the use of TaqMan real-time PCR as a sensitive technique to measure and quantify plasma HIV-1 RNA or proviral HIV-1 DNA (Lewin et al, 1999;de Baar et al, 2001;Desire et al, 2001;Hance et al, 2001;Zhao et al, 2002). However, TaqMan real-time PCR has not been used to detect two different HIV-1 primary isolates in growth competition experiments in order to estimate virus fitness.…”

Detection of HIV-1 P24 Gag in Plasma by a Nanoparticle-Based Bio-Barcode-Amplification Method

“…Detection of the major core protein of HIV-1 (p24 Gag) in serum or plasma by an ELISA is specific but does not offer the requisite sensitivity [5,6,[18][19][20][21]. Other methods for assessing the presence of virus include PCR-based detection of the viral genome, specific primers and probes in plasma and viral co-culture with normal donor lymphocytes [3,7,8,16]. Although accurate, these techniques are time-consuming and require specialized laboratories, elaborate equipment, special specimen-transport conditions to maintain viability and highly skilled personnel.…”

“…Keywords bio-barcode-amplification method; HIV-1; major core protein of HIV-1 (p24 Gag) Accurate diagnostic tests for HIV-1 have a key role in clinical care and the control of HIV/ AIDS [1][2][3][4][5][6]. Commonly used methods rely on the detection of antibodies to HIV-1 in plasma or sera [2,[7][8][9].…”

Development, Evaluation, and Validation of an Oligonucleotide Probe Hybridization Assay To Subtype Human Immunodeficiency Virus Type 1 Circulating Recombinant Form CRF02_AG

“…This assay was configured to distinguish CRF02_AG from all other subtypes and CRFs. The close genetic distance between subtype A fragments in CRF02_AG and subtype A strains hampered differentiation between CRF02_AG and subtype A by env HMAs (7) as well as by a one-tube real-time isothermal amplification subtyping method described by de Baar et al (6). A DNA enzyme immunoassay genotyping method for the env gene developed by Plantier et al (22) showed a sensitivity of 86.6% (13 of 15 samples) for identifying CRF02_AG infections.…”