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Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR.

ABSTRACT: BACKGROUND: In real-time PCR data analysis, the cycle threshold (CT) method is currently the gold standard. This method is based on an assumption of equal PCR efficiency in all reactions, and precision may suffer if this condition is not met. Nonlinear regression analysis (NLR) or curve fitting has therefore been suggested as an alternative to the cycle threshold method for absolute quantitation. The advantages of NLR are that the individual sample efficiency is simulated by the model and that absolute quantitation is possible without a standard curve, releasing reaction wells for unknown samples. However, the calculation method has not been evaluated systematically and has not previously been applied to a TaqMan platform. AIM: To develop and evaluate an automated NLR algorithm capable of generating batch production regression analysis. RESULTS: Total RNA samples extracted from human gastric mucosa were reverse transcribed and analysed for TNFA, IL18 and ACTB by TaqMan real-time PCR. Fluorescence data were analysed by the regular CT method with a standard curve, and by NLR with a positive control for conversion of fluorescence intensity to copy number, and for this purpose an automated algorithm was written in SPSS syntax. Eleven separate regression models were tested, and the output data was subjected to Altman-Bland analysis. The Altman-Bland analysis showed that the best regression model yielded quantitative data with an intra-assay variation of 58% vs. 24% for the CT derived copy numbers, and with a mean inter-method deviation of x 0.8. CONCLUSION: NLR can be automated for batch production analysis, but the CT method is more precise for absolute quantitation in the present setting. The observed inter-method deviation is an indication that assessment of the fluorescence conversion factor used in the regression method can be improved. However, the versatility depends on the level of precision required, and in some settings the increased cost effectiveness of NLR may justify the lower precision.

SUBMITTER: Goll R

PROVIDER: S-EPMC1450306 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR.

Goll Rasmus R Olsen Trine T Cui Guanglin G Florholmen Jon J

BMC bioinformatics 20060303

<h4>Background</h4>In real-time PCR data analysis, the cycle threshold (CT) method is currently the gold standard. This method is based on an assumption of equal PCR efficiency in all reactions, and precision may suffer if this condition is not met. Nonlinear regression analysis (NLR) or curve fitting has therefore been suggested as an alternative to the cycle threshold method for absolute quantitation. The advantages of NLR are that the individual sample efficiency is simulated by the model and ...[more]

PMID: 16515700

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Project description:BackgroundChandipura virus (CHPV), a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55-75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.MethodsPrimers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice) in ovo (eggs), in vitro (Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.ResultsReal-time one step RT-PCR was optimized using in vitro transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 x 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 x 102 PFU/ml). Vero and PS cell-lines (1.2 x 103 PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.ConclusionOn account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.

Dataset Information

Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR.

Publications

Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR.

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