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- Title
Clinical validation of a real-time PCRfor HLA typing of Coeliac disease.
- Authors
Toogood, Roger; Ogbuigwe, Paul; Halauafu, Taukei
- Abstract
Objectives: Coeliac disease has a studied relationship with the HLA alleles DQB*02 and DQB*03:02, with the absence of both allowing Coeliac disease to be ruled out during diagnosis. Validation of a high-throughput qualitative real-time PCR will allow for fast and sensitive screening of patients for these HLA markers. Our objective is to validate this real-time PCR for clinical use on a new analyser, transitioning it from an outdated system. Methods: A qualitative real-time PCR using SYBR green melting curve analysis was performed for 53 patient samples. Each was run on the previously validated Roche LightCycler 2.0 and the Roche LightCycler 480 using separate probes and cycles conditions for the two alleles. A dilution series of positive controls was performed to compare the limits of detection for the PCR on both analysers. Results: The Roche LightCycler 480 was found to have the same limit of detection and level of sensitivity as the previously validated Roche LightCycler 2.0 method using the same PCR programme and reagents. The PCR showed 100% sensitivity and specificity in detection of HLA alleles DQB*02 and DQB*03:02. Conclusion: Transition of the PCR to the Roche LightCycler 480 will not result in a decrease in sensitivity, specificity or limit of detection compared to the Roche LightCycler 2.0 and is suitable for clinical use. In addition, the number of samples that can be handled in a single run is increased from 36, on the LightCycler 2.0, to 96 on the LightCycler 480. Thereby reducing turnaround times and making the workflow more efficient.
- Subjects
CELIAC disease diagnosis; HLA-B27 antigen; PREDICTIVE tests; ALLELES; CELIAC disease; WORKFLOW; RISK assessment; DESCRIPTIVE statistics; POLYMERASE chain reaction; SENSITIVITY &; specificity (Statistics); DISEASE risk factors
- Publication
New Zealand Journal of Medical Laboratory Science, 2023, Vol 77, Issue 2, p99
- ISSN
1171-0195
- Publication type
Article