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Objectives: The Spartan DX^TM (Spartan Bioscience Inc.; Ottawa, ON) is a compact, inexpensive, 4-well real-time PCR (RTPCR) instrument. It uses a 2-temperature reaction cycle that promises more rapid results than conventional RTPCR. The diagnosis of Herpes Simplex Virus (HSV) infections of the Central Nervous System (CNS) is a logical application for this instrument: on-demand, non-batched analysis where speed and convenience are critical. We evaluate the performance of a TaqMan®-based HSV assay adapted for the Spartan DX.
Methods: The RTPCR HSV assay used at Children’s Hospital of Eastern Ontario (CHEO) (“reference-HSV”) was modified for the Spartan DX (“Spartan-HSV”). Replicate testing of Cerebral Spinal Fluid (CSF) spiked with HSV-1 and with HSV-2 DNA (180 copies and 160 copies, respectively; ABi Inc., Columbia, MD) was performed. HSV-1 stock (3.2 TCID50; 1 log > the lower limit of detection of reference-HSV) was added to blank CSF, extracted, and tested in same- and separate-day runs. Finally, archived CSF specimens (23 PCR [+] and 10 PCR [-]) were tested. Extractions were performed with the QIAmp DNA Mini Kit. Specimens were tested in parallel by reference-HSV (ABI 7500 or BioRad iCycler). Spartan- and reference-HSV results (PCR [+/-], CT) were compared.
Results: Spartan-HSV detected both HSV-1 and HSV-2 DNA: the mean CT values of samples spiked with 180 copies HSV-1/reaction (n=8) and with 160 copies HSV-2/reaction (n=8), were 35 (range: 34-36). For CSF samples spiked with 3.2 TCID50 HSV-1 (n=46), the mean CT value was 36.6 (range: 35-39). For all study samples, Spartan and reference test results were in agreement. For clinical specimens, there was no meaningful CT value difference between methods (Spartan CT = reference +/-2 CT).
Discussion/Conclusion: The Spartan-HSV assay is reliable and reproducible, with performance equaling conventional RT HSV PCR. The Spartan DX is an excellent choice for laboratories needing an accurate, affordable, convenient platform to accommodate non-batched “stat” testing, or for smaller laboratories seeking to implement real-time molecular diagnostics in their facilities.

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Objectives: Identification of vancomycin-resistant enterococci (VRE) is important to limit nosocomial spread and has traditionally relied on phenotypic methods. A rapid real-time PCR assay detecting vanA, vanB or vanC resistance genes was developed for use on a new instrument using a new probe technology. The Spartan DX™ is a singleplex real-time PCR instrument, with four wells, designed for on-demand, non-batched applications where speed and convenience are important. BHQplus™ is a probe chemistry that increases probe melting temperature and enables more sensitive fluorescence detection. The purpose of this study was to evaluate the Spartan DX and BHQplus probes to detect and genotype VRE isolates.
Methods: Three sets of primers with corresponding BHQplus TaqMan® probes or regular TaqMan probes were designed against the same conserved regions of the vanA, vanB, vanB2/3, vanC1 and vanC2/C3 genes. To date, the isolates tested include 26 enterococci with either vanA or vanB, 7 with vanC and 10 non-enterococci (including S. aureus, E. coli, coag-negative Staphylococcus, group C and G Streptococcus). Amplification was performed from crude DNA lysates. Real-time PCR was performed using both regular TaqMan probes and BHQplus TaqMan probes to vanA, vanB and vanC, to genotype the isolates. The specificity and efficiency of the assay was validated by performing serial dilutions and examining cross-reactivity by PCR and gel electrophoresis.
Results: Our preliminary results indicate that BHQplus probes specifically identified vanA, vanB, vanB2/B3, vanC1 and vanC2/C3 genes from all tested enterococcus isolates without cross-reactivity between the van genes. The regular TaqMan probes only recognized the vanA, vanB and vanB2/B3 genes. Efficient real-time amplification was detected with serially diluted samples down to the 1-10 copy range. In addition, none of the VRE BHQplus primer sets cross-reacted with non-enterococcus lysates tested.
Conclusion: These preliminary results demonstrate specific and rapid identification of vanA, vanB or vanC genes in enterococci from crude lysates. Use of BHQplus probes results in increased reaction sensitivity, and allowed for the creation of a more effective and efficient real-time PCR assay.