Real-time PCR probe optimization using design of experiments approach

Wadle, Simon; Lehnert, Michael; Rubenwolf, Stefanie; Zengerle, Roland; Stetten, Felix von

doi:10.1016/j.bdq.2015.12.002

Cited by 25 publications

(23 citation statements)

References 30 publications

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“…MP PCR showed significantly lower LoDs than HP PCR (e.g., for the mecA resistance gene, the MP was 18 copies per reaction compared to 99 copies per reaction for HP). This is primarily due to a higher SNR when using the URs for signal generation compared with the dual-labeled HPs, as shown previously (9). The higher SNR is in turn a function of the 23%99% reduced fluorescence background of URs compared with HPs (Supplementary Material).…”

Section: Resultsmentioning

confidence: 71%

“…Readout was performed as described for the food panel reactions. LoDs were determined using SPSS software version 19 probit analysis (IBM, Armonk, NY) (17) with conditions for positive amplifications as described before (9). …”

Section: Methodsmentioning

confidence: 99%

“…Mediator probe (MP) PCR is an example of USD (8). Design rules have been published recently for both single and biplex MP PCR (9,10). These rules comprise the design of the label-free target-specific MPs, which are cleaved during primer extension, as well as the design of the universal reporter oligonucleotides (URs).…”

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confidence: 99%

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Simplified Development of Multiplex Real-Time PCR Through Master Mix Augmented by Universal Fluorogenic Reporters

et al. 2016

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Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.

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Section: Resultsmentioning

confidence: 71%

Section: Methodsmentioning

confidence: 99%

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Simplified Development of Multiplex Real-Time PCR Through Master Mix Augmented by Universal Fluorogenic Reporters

et al. 2016

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“…It is very interesting that the multiplex RT-PCR developed in the present study was capable of detecting a single pathogen from a clinical sample, and could be amplified more than one pathogen in the co-infections. The multiplex RT-PCR was clearly capable of detecting more than 2 pathogens, simultaneously (33). Successful amplification of all 3 templates in the same reaction mixture with a clinical sample required additional Taq polymerase in the multiplex reaction, because at a limited concentration of Tag, different reaction mixtures containing different copy numbers of various templates and different separate amplicons were not observed (34).…”

Section: Discussionmentioning

confidence: 99%

Development of Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Detection of Influenza A, B and Adenoviruses

et al. 2018

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Polymerase chain reactionn Influenza A Influenza B Adenovirus Multiplex RT-PCR Respiratory infection Background and objective: Millions of people in developing countries lose their lives due to acute respiratory infections, such as Influenza A & B and Adeno viruses. Given the importance of rapid identification of the virus, in this study the researchers attempted to design a method that enables detection of influenza A, B, and adenoviruses, quickly and simultaneously. The Multiplex RT PCR method was the preferred method for the detection of influenza A, B, and adenoviruses in clinical specimens because it is rapid, sensitive, specific, and more cost-effective than alternative methods Methods: After collecting samples from patients with respiratory disease, virus genome was extracted, then Monoplex PCR was used on positive samples and Multiplex RT-PCR on clinical specimens. Finally, by comparing the bands of these samples, the type of virus in the clinical samples was determined. Results: Performing Multiplex RT-PCR on 50 samples of respiratory tract led to following results; flu A: 12.5%, fluB: 50%, adeno: 27.5%, negative: 7.5%, and 2.5% contamination. Conclusion: Reverse transcription-multiplex Polymerase Chain Reaction (PCR) technique, a rapid diagnostic tool, has potential for high-throughput testing. This method has a significant advantage, which provides simultaneous amplification of numerous viruses in a single reaction. This study concentrates on multiplex molecular technologies and their clinical application for the detection and quantification of respiratory pathogens. The improvement in diagnostic testing for viral respiratory pathogens effects patient management, and leads to more costeffective delivery of care. It limits unnecessary antibiotic use and improves clinical management by use of suitable treatment.

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“…We think that our primer sets can be widely used for applications such as qPCR and digital PCR to investigate changes in gene expression and donor variability. However, method validation based on common guidelines for PCR assay optimization is crucial for new PCR applications [32].…”

Section: Discussionmentioning

confidence: 99%

Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

George

Rödiger

Schröder

et al. 2016

JCB

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Abstract.Severe heart diseases such as myocarditis and cardiomyopathy are often characterized by progressive damages of contractile heart tissue which ultimately can lead to terminal heart failure. There is a need for relevant in vitro cultures of human cardiomyocytes to study pathogenic processes and to perform pharmacological testing of new heart drugs. By using the upcyte/EPCC (enhanced primary cell culture) approach for direct multiplication of organ-specific cells, we established proliferating human cardiomyocyte cultures derived from atrial appendages. For qualitative cardiac expression profiling we established a comprehensive set of multiplex PCR assays, selected from a panel of 32 genes, to rapidly screen changes at the transcriptional level in human ventricular and atrial cardiomyocytes. Our multiplex PCR approach revealed some donor variability of native atrial heart tissue that need to be confirmed by further studies with more samples. Our initial studies further indicated that characteristic heart muscle cell markers such as MLC-2a, MLC-2v, CHRM2, ADRB1, DES, EDRNB, Cx40 and KCNA5 were down-regulated when isolated cardiomyocytes were taken into primary cell culture. Compared to native heart tissue, proliferating atrial cardiomyocytes lacked expression of those cardiac markers but still expressed MYCD, GATA-4, Cx43, SERCA2, BNP, Tbx5, EDNRA and ACTB. Surprisingly, atrium-derived cardiomyocytes started to express NFAc4 in passage three, and cardiomyocyte marker expressions of Cx43 and BNP were even increased over cultivation time.In conclusion, our novel multiplex PCR assays should be useful for expression profiling of native heart tissues from patients with different disease conditions and for characterization of in vitro cardiomyocyte cultures.Keywords: Atrial appendages, ACTA1, ACTB ACTC1, ADRB1, ADRB2, ATP2A2, atrial fibrillation, BMP2, CHRM2, cluster analysis, DES, EDNRA, EDNRB, enhanced primary cell cultures (EPCC), GAPDH, GATA4, gene expression, GJA1, GJA5, HPRT1, KCNA5, KCNH2, multiplex PCR, MYL2, MYL7, MYOCD, NFATC4, NKX2-5, NPPA, NPPA, PLN, qPCR, reference genes, RPLP0, SCN5A, TBX5, TNNI3, TNNT2, upcyte genes, VIM Abbreviations EPCC enhanced primary cell cultures HE hematoxylin -eosin qPCR quantitative PCR 1 These authors contributed equally.

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Real-time PCR probe optimization using design of experiments approach

Abstract: Graphical abstract

Cited by 25 publications

References 30 publications

Simplified Development of Multiplex Real-Time PCR Through Master Mix Augmented by Universal Fluorogenic Reporters

Simplified Development of Multiplex Real-Time PCR Through Master Mix Augmented by Universal Fluorogenic Reporters

Development of Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Detection of Influenza A, B and Adenoviruses

Development of multiplex PCR systems for expression profiling of human cardiomyocytes induced to proliferate by lentivirus transduction of upcyte genes

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