The importance of set up time and temperature in real—time PCR; an essential reminder

Cassidy, Hayley; Maclean, Alasdair; Gunson, Rory

doi:10.1016/j.jviromet.2017.02.005

Cited by 2 publications

(1 citation statement)

References 2 publications

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“…65 Thus, in a 2022 comparison of 12 African swine fever virus commercial PCRs, mean Cq values were in general agreement. 45 Standardization is crucial to reliable test performance, but normalization is needed to control for the run-to-run variation arising from differences in samples 19,61,66,68 and in sample handling and processing, 9,24,25,67 as well as from differences among technicians 20 and among manufacturing lots. 6 For ELISAs, normalization was achieved by expressing the sample response (i.e., absorbance values) relative to positive and/or negative reference sera run on the same plate (i.e., sample:positive or sample:negative ratios).…”

Section: Discussionmentioning

confidence: 99%

Normalizing real-time PCR results in routine testing

Armenta-Leyva,

Munguía-Ramírez,

Cheng

et al. 2023

J VET Diagn Invest

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Normalization, the process of controlling for normal variation in sampling and testing, can be achieved in real-time PCR assays by converting sample quantification cycles (Cqs) to “efficiency standardized Cqs” (ECqs). We calculated ECqs as E−ΔCq, where E is amplification efficiency and ΔCq is the difference between sample and reference standard Cqs. To apply this approach to a commercial porcine reproductive and respiratory syndrome virus (PRRSV) RT-qPCR assay, we created reference standards by rehydrating and then diluting (1 × 10−4) a PRRSV modified-live vaccine (PRRS MLV; Ingelvac) with serum or oral fluid (OF) to match the sample matrix to be tested. Sample ECqs were calculated using the mean E and reference standard Cq calculated from the 4 reference standards on each plate. Serum ( n = 132) and OF ( n = 130) samples were collected from each of 12 pigs vaccinated with a PRRSV MLV from −7 to 42 d post-vaccination, tested, and sample Cqs converted to ECqs. Mean plate Es were 1.75–2.6 for serum and 1.7–2.3 for OF. Mean plate reference standard Cqs were 29.1–31.3 for serum and 29.2–31.5 for OFs. Receiver operating characteristic analysis calculated the area under the curve for serum and OF sample ECqs as 0.999 (95% CI: 0.997, 1.000) and 0.947 (0.890, 1.000), respectively. For serum, diagnostic sensitivity and specificity of the commercial PRRSV RT-qPCR assay were estimated as 97.9% and 100% at an ECq cutoff ≥ 0.20, and for OF, 82.6% and 100%, respectively, at an ECq cutoff ≥ 0.45.

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