Statistical significance of quantitative PCR

Karlen, Yann; McNair, Alan; Perseguers, Sébastien; Mazza, Christian; Mermod, Nicolas

doi:10.1186/1471-2105-8-131

Cited by 318 publications

(282 citation statements)

References 37 publications

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“…Real-time quantitative PCR was performed using fluorescence dye SYBR green I, and each sample was analyzed in triplicate as described previously. 50 The following primers were used: GFP (forward, 5¢-ACATTATGCCGGACAAAGCC-3¢; reverse, 5¢-TTGTTT GGTAATGATCAGCAAGTTG-3¢) and the housekeeping GAPDH gene (forward primer, 5¢-CGACCCCTTCATTGACCTC-3¢; reverse primer, 5¢-CTCCACGACA TACTCAGCACC-3¢). The number of GFP transgenes integrated for each clone was estimated using the conserved housekeeping gene GAPDH as an internal reference.…”

Section: Generation Of Seap Producer Cellsmentioning

confidence: 99%

Use of human MAR elements to improve retroviral vector production

et al. 2010

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Section: Generation Of Seap Producer Cellsmentioning

confidence: 99%

Use of human MAR elements to improve retroviral vector production

et al. 2010

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“…The presence of two copies of the primer-specific PinsEXO1 sequence in the P. insidiosum genome increased the chances of successful PCR amplification. The E value of 108% (acceptable range 90-110%) and the R 2 value of 0.994 (acceptable range w0.990) confirmed that this assay is highly efficient, and that the relationship between C q and DNA quantity is linear (Cankar et al, 2006;Karlen et al, 2007). The intra-assay (1.5-3.1%) and inter-assay (1.5%) CVs of the RT-PCR were relatively low (acceptable range v10%), reflecting the reproducibility of the assay (Cankar et al, 2006;Karlen et al, 2007).…”

Section: Discussionmentioning

confidence: 75%

“…The E value of 108% (acceptable range 90-110%) and the R 2 value of 0.994 (acceptable range w0.990) confirmed that this assay is highly efficient, and that the relationship between C q and DNA quantity is linear (Cankar et al, 2006;Karlen et al, 2007). The intra-assay (1.5-3.1%) and inter-assay (1.5%) CVs of the RT-PCR were relatively low (acceptable range v10%), reflecting the reproducibility of the assay (Cankar et al, 2006;Karlen et al, 2007). Taken together, the RT-PCR of PinsEXO1 was successfully developed for detecting and quantifying gDNA of P. insidiosum.…”

Section: Discussionmentioning

confidence: 75%

Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-β-glucanase

Keeratijarut

Lohnoo

Yingyong

et al. 2015

Journal of Medical Microbiology

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Pythiosis is a life-threatening infectious disease caused by Pythium insidiosum. Early and accurate diagnosis is the key to prompt treatment and an improved prognosis for patients with pythiosis. An alternative to microbiological and immunological approaches for facilitating diagnosis of pythiosis is the PCR-based assay. Until recently, the ribosomal DNA (rDNA) region was the only target available for PCR-based detection of P. insidiosum. Failure to detect P. insidiosum by PCR amplification using the rDNA-specific primers has been reported. PinsEXO1, encoding an exo-1,3-b-glucanase, is an alternative, novel and efficient target for identification of P. insidiosum by conventional PCR. In this study, we aimed to develop a realtime (RT)-PCR approach targeting PinsEXO1 and compare its performance with conventional PCR for the detection of P. insidiosum. Both conventional and RT-PCR assays were positive for all 35 P. insidiosum strains tested, whilst all 58 control fungi were negative. The turnaround time for conventional PCR was 10 h, whilst that for RT-PCR was 7.5 h. The lowest amounts of genomic DNA template required for successful amplification by conventional and RT-PCR were 1 and 1610 24 ng, respectively. In conclusion, the RT-PCR assay retained 100 % sensitivity and 100 % specificity for detection of P. insidiosum. It showed a substantially improved analytical sensitivity and turnaround time that could improve diagnosis of pythiosis. The assay could also facilitate quantitative DNA analysis and epidemiological studies of P. insidiosum.

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“…While the PDV and GAPDH singleplex reactions had excellent precision, there was a slight loss of precision and PCR efficiency in duplex. Efficiency over 110% could suggest possible overamplification of the more abundant target in the duplex reaction or a decrease in the theoretical doubling of DNA in reaction amplification at the far ends of the range of detection (Bio-Rad Laboratories 2006; Applied Biosystems 2007; Karlen et al 2007). The kinetics related to the amplification of plasmid-derived RNA also differ from tissue-derived RNA as well as ds-DNA plasmid standards (Plaffl 2004).…”

Section: Discussionmentioning

confidence: 99%

DEVELOPMENT OF A ONE-STEP DUPLEX RT-qPCR FOR THE QUANTIFICATION OF PHOCINE DISTEMPER VIRUS

Bogomolni

Frasca

Matassa

et al. 2015

Journal of Wildlife Diseases

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ABSTRACT:Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptasequantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 10 0 to 10 9 copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 10 0 to 10 9 copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.

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Statistical significance of quantitative PCR

Cited by 318 publications

References 37 publications

Use of human MAR elements to improve retroviral vector production

Use of human MAR elements to improve retroviral vector production

Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-β-glucanase

DEVELOPMENT OF A ONE-STEP DUPLEX RT-qPCR FOR THE QUANTIFICATION OF PHOCINE DISTEMPER VIRUS

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