Background:  A rapid, simple, cost-efficient nucleic acid–based test for detecting HIV in resource-poor or field settings is highly desirable. Loop-mediated isothermal amplification is a novel method for amplifying DNA and RNA (LAMP and RT-LAMP, respectively) with high specificity, sensitivity, and simplicity. The method consists of incubating a mixture of the target gene, 4 or 6 different primers, Bst DNA polymerase, AMV reverse transcriptase (for RNA), and substrates. This is a 1-step, isothermal reaction with a rapid outcome and no expensive equipment. In addition, increased turbidity from the formation of the by-product magnesium pyrophosphate, permits visual detection. This makes the LAMP method an attractive alternative for traditional polymerase chain reaction (PCR), which requires dedicated equipment and can be time-consuming. For the first time, the LAMP and RT-LAMP methods have been applied for detection of HIV-1 and their sensitivity, specificity, and applicability for clinical specimens have been evaluated.

Methods:  Primers targeted against highly conserved regions of HIV-1 were designed using software found on the Eiken Web page. For initial proof of concept, DNA was extracted from OM10.1 cells and RNA from a commercial source of Bal virus. Subsequently, plasma from HIV-1-infected individuals, with known viral loads, was used either directly after heat treatment (99^ºC for 10 minutes) or after RNA extraction for amplification. To address specificity, HIV-negative and HTLV-I-infected samples were used. A range of incubation times and temperatures were examined to determine optimum amplification conditions.  

Results:  As early as 30 minutes at 60^ºC, 10² HIV-1 proviral copies were detected. For extracted RNA, detection was in the range of 10³ viral copies. Furthermore, we were able to amplify from as little as 5 µL of heat-treated plasma, with no RNA extraction. No amplification occurred using DNA, RNA, or plasma from HTLV-I-infected or HIV-negative samples.

Conclusions:  The HIV LAMP or RT-LAMP assay is a cost-effective, single-tube assay that requires no sophisticated equipment. Its simplicity gives the assay potential as a diagnostic tool. The ability to amplify without extraction of nucleic acids, lower cost, and the rapid nature of the assay make this method highly attractive for use in resource-poor settings and field applications.