Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high falsenegative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity. (J Mol

Summary There is a clear need for an instrument-free molecular diagnostic system for detecting HIV-1 RNA or DNA that can be used in developing countries. Such a test could find utility in the early diagnosis of HIV-1 infection in infancy, as well as serve as a surrogate end-point for vaccine trials. We have developed an IsoAmp® HIV-1 assay (BioHelix Corp, Beverly, MA) targeting the HIV-1 gag gene using our isothermal reverse transcription helicase dependent amplification chemistry (RT-HDA). This IsoAmp® HIV assay uses a disposable amplicon containment device with an embedded vertical-flow DNA detection strip to detect the presence of HIV-1 amplicons. The vertical-flow DNA detection strip has a control line to validate the performance of the device as well as a test line to detect the analyte. The analyte is detected by a sandwich immunoassay for reporter moieties on a capture probe and a detection probe. The control line consists of the detection probe reporter moiety conjugated to the vertical-flow DNA detection strip. The preliminary limit of detection of the IsoAmp HIV assay was evaluated by testing serial dilutions of the HIV-1 Armored® RNA (Assuragen, Austin TX). We found that twenty-one out of twenty-eight (75%) assays were positive when 50 copies of the HIV-1 Armored RNA were input into the IsoAmp HIV reaction.

In vitro DNA amplification methods, such as polymerase chain reaction (PCR), rely on synthetic oligonucleotide primers for initiation of the reaction. In vivo, primers are synthesized on-template by DNA primase. The bacteriophage T7 gene 4 protein (gp4) has both primase and helicase activities. In this study, we report the development of a primase-based Whole Genome Amplification (pWGA) method, which utilizes gp4 primase to synthesize primers, eliminating the requirement of adding synthetic primers. Typical yield of pWGA from 1 ng to 10 ng of human genomic DNA input is in the microgram range, reaching over a thousand-fold amplification after 1 h of incubation at 37°C. The amplification bias on human genomic DNA is 6.3-fold among 20 loci on different chromosomes. In addition to amplifying total genomic DNA, pWGA can also be used for detection and quantification of contaminant DNA in a sample when combined with a fluorescent reporter dye. When circular DNA is used as template in pWGA, 108-fold of amplification is observed from as low as 100 copies of input. The high efficiency of pWGA in amplifying circular DNA makes it a potential tool in diagnosis and genotyping of circular human DNA viruses such as human papillomavirus (HPV).