Biomedical subjects

David Hillyard

Publications and source records attributed to David Hillyard.

4 recordsLinked to original sources

PubMed2007

Use of modified oligonucleotides to compensate for sequence polymorphisms in the real-time detection of norovirus.

Noroviruses are major causative agents of sporadic and outbreak-associated nonbacterial gastroenteritis worldwide. Real-time RT-PCR has rapidly become the principle means for norovirus detection due to its sensitivity and specificity; however sequence variations in the Norovirus genome can cause problems for real-time chemistries. Using a combination of modified bases and minor groove binder-conjugated Eclipse hybridization probes, a one step real-time RT-PCR assay was designed and optimized to detect norovirus genogroups I and II. Additionally, an RNA internal control was incorporated into the assay to monitor nucleic acid extraction and amplification inhibition. As part of this study, a combination of 36 stool and RNA samples were tested and results were compared to a previously described TaqMan assay. An overall correlation of 97% was obtained. After 7 months of clinical testing, a percent positive rate of 39% was determined, with genogroup II accounting for 98% of the positives. Overall, the Eclipse assay is a sensitive and robust method for detecting and typing norovirus genogroups I and II as well as useful for routine clinical testing. Incorporating modified bases helps overcome design limitations due to single nucleotide polymorphisms.

Base Sequence↗

PubMed2005

Use of lyophilized standards for the calibration of a newly developed real time PCR assay for human herpes type six (HHV6) variants A and B.

A quantitative real time PCR assay utilizing an Eclipse minor groove binding hybridization probe was developed to detect and type human herpes 6. A 115 base pair product from the U67 gene was selected for amplification and the assay included a noncompetitive internal control. The probe's melting temperature from the amplified sequence differentiated between HHV6 variants (A and B). In this study, 120 samples (60 spiked and 60 negative) comprising CSF, plasma, and serum were tested at high and medium levels, and near the limit of quantitation. The use of stored standard curves for assay calibration was compared to curves run on each assay, and the stability of liquid frozen versus lyophilized frozen stocks for calibrators and controls was assessed. After 9 months of clinical testing, assay performance was examined to determine the percent positive rate and positive sample reproducibility, as well as to evaluate standard curve stability. We obtained 100% correlation to expected results for positive and negative samples. A stored curve proved easier, more cost effective, and more reliable than running a standard curve on each assay. The use of lyophilized standards contributes substantially to the maintenance of reproducible testing over an extended period of time.

Blood↗

PubMed2005

Effect of sequence polymorphisms on performance of two real-time PCR assays for detection of herpes simplex virus.

Herpes simplex virus (HSV) is the most common cause of acquired, sporadic encephalitis in the United States. PCR identification of HSV in spinal fluid has become the diagnostic gold standard due to its sensitivity and potential for speed, replacing other methods such as culture. We developed a real-time PCR assay to detect HSV, using a new type of hybridization probe, the Eclipse probe. In this study, we ran 323 samples (171 positives and 152 negatives) with the Eclipse real-time PCR assay and compared these results with another PCR assay using gel detection. The real-time assay agreed with our reference method for 319 out of the 323 samples tested (99%). Using two different real-time PCR platforms, we discovered that SNPs within the amplicon's probe binding region that are used to distinguish HSV-1 from HSV-2 can decrease assay sensitivity. This problem is potentially a general one for assays using fluorescent probes to detect target amplification in a real-time format. While real-time PCR can be a powerful tool in the field of infectious disease, careful sequence evaluation and clinical validation are essential in creating an effective assay.

Base Sequence↗

PubMed2004

Hepatitis C genotyping by denaturing high-performance liquid chromatography.

Determination of the hepatitis C virus (HCV) genotype for infected patients increasingly has become accepted as the standard of care. Genotype assignment helps in assessing disease prognosis and assists in establishing the appropriate duration of treatment. The great genetic diversity of HCV, with 11 major genotypes and >70 subtypes, contributes to the technical difficulty of genotype testing. While the "gold standard" for testing is nucleic acid sequencing, a variety of hybridization assays, including the line probe assay, have been developed to provide more rapid and accessible forms of testing. The aim of this study was to determine whether denaturing high-performance liquid chromatography (dHPLC) could be used as a clinical method for distinguishing HCV genotypes 1, 2, 3, and 4. A portion of the 5' untranslated region of the HCV genome was amplified by heminested multiplex reverse transcription PCR. The two amplicons then were analyzed by dHPLC analysis and compared to the genotypes determined by sequence analysis. After 115 specimens were analyzed as standards, 200 masked specimens (specimens whose identity was not known before testing) were analyzed to determine the concordance of the assay. The assay had a concordance of 96% at the genotype level and a concordance of 87% at the subtype level. However, the dHPLC method was not as accurate as other reported methods of HCV genotyping. This is the first time that HCV genotyping has been performed by dHPLC.

5' Untranslated Regions↗