Comparative evaluation of the Seegene Seeplex RV15 and real-time PCR for respiratory virus detection.

Respiratory virus infections contribute substantially both to hospitalizations of young children, and to morbidity in immunocompromised patients such as those with hematological malignancies. Their rapid and accurate diagnosis is essential to patient management. To evaluate the prospective utility of Seeplex® DPO technology in respiratory virus diagnosis, a panel of 99 respiratory samples positive by real-time RT-PCR for one or more viruses was assayed by the Seegene Seeplex® RV12 system. As well as being able to detect all 10 viruses in the real-time RT-PCR system with the exception of enteroviruses, RV12 can also distinguish between the two subgroups of RSV and detect two subgroups of coronaviruses. Seven of the nine viruses in common with the RT-PCR were detected reliably by RV12. Eleven samples RT-PCR-positive for Metapneumovirus and five samples positive for influenza B were not detected by RV12. Seegene developed a second-generation system, RV15, which not only allowed detection of three additional viruses, but also addressed the potential problems with RV12 specificity. To address these concerns, 84 respiratory samples positive for a range of viruses by real-time PCR were assayed with RV15. The results of this evaluation improved significantly upon those seen with RV12. The high throughput capabilities and potential lower technical requirements afforded by the Seeplex® system may offer an alternative to real-time RT-PCR systems.

Development of a loop-mediated isothermal amplification (LAMP) assay for the sensitive detection of Haemonchus contortus eggs in ovine faecal samples.

A major constraint on the effective control and management of helminth parasites in livestock is the lack of rapid and reliable diagnostic tests to identify the parasite species responsible for disease and to allow informed treatment decisions to be made. In the present study, we have developed a novel DNA-based assay for the detection of Haemonchus contortus eggs in ovine faecal samples, using loop-mediated isothermal amplification (or LAMP). LAMP allows for rapid detection of H. contortus DNA under isothermal incubation conditions. The robust nature of this assay negates the need for extensive DNA extraction, allowing amplification from relatively crude samples. Preliminary results suggest that LAMP is highly specific, and does not cross-react with DNA from other common co-infecting parasites. The Haemonchus LAMP assay is also highly sensitive, exhibiting a 10 times lower detection limit than the equivalent PCR; 10(-5) ng/µl and 10(-4) ng/µl DNA, respectively, allowing detection in a faecal samples containing two Haemonchus eggs per gram. Translation of this assay onto a real-time platform provided rapid results and highlighted its potential as a quantitative assay which could inform treatment decisions in the future.