Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

Mackay, Ian M.; Arden, Katherine E.; Nitsche, Andreas

doi:10.1016/s0580-9517(04)34010-9

Cited by 9 publications

(9 citation statements)

References 461 publications

(494 reference statements)

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“…With the increasing number of genomic sequences deposited in public databases like GISAID, the primers and probes applied for SARS-CoV-2 detection in PCR 1 and PCR 2 were subjected to controls by aligning the oligonucleotide sequences to full genome alignments of all available SARS-CoV-2 genome sequences. Common PCR design rules were applied to assess the validity of the PCR assays [ 11 , 12 ].…”

Section: Methodsmentioning

confidence: 99%

Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

Michel

Neumann

Krause

et al. 2021

Virol J

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Background The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. Aim Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). Methods Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. Results Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. Conclusion The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.

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Section: Methodsmentioning

confidence: 99%

Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

Michel

Neumann

Krause

et al. 2021

Virol J

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“…To address this problem, more specific real-time detection methods use labeled primers for incorporation into the amplicon, e.g. Sunrise primers [2] or Scorpion primers [3], or add additional probes to the reactions that hybridize to the amplicon in a sequence-dependent manner [4], [5]. Such DNA probes commonly carry a fluorescent label and a quencher for background control, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…Molecular Beacons are not digested during PCR, and therefore allow for melting curve analysis after the PCR amplification has been completed. Many other approaches to real-time PCR and fluorescent probes have been described in the literature [4], [5], [10], [11] following similar principles as outlined above for the most commonly used tools.…”

Section: Introductionmentioning

confidence: 99%

Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

Hanami¹,

Delobel²,

Kanamori³

et al. 2013

PLoS ONE

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Real-time monitoring of PCR is one of the most important methods for DNA and RNA detection widely used in research and medical diagnostics. Here we describe a new approach for combined real-time PCR monitoring and melting curve analysis using a 3′ end-blocked Exciton-Controlled Hybridization-sensitive fluorescent Oligonucleotide (ECHO) called Eprobe. Eprobes contain two dye moieties attached to the same nucleotide and their fluorescent signal is strongly suppressed as single-stranded oligonucleotides by an excitonic interaction between the dyes. Upon hybridization to a complementary DNA strand, the dyes are separated and intercalate into the double-strand leading to strong fluorescence signals. Intercalation of dyes can further stabilize the DNA/DNA hybrid and increase the melting temperature compared to standard DNA oligonucleotides. Eprobes allow for specific real-time monitoring of amplification reactions by hybridizing to the amplicon in a sequence-dependent manner. Similarly, Eprobes allow for analysis of reaction products by melting curve analysis. The function of different Eprobes was studied using the L858R mutation in the human epidermal growth factor receptor (EGFR) gene, and multiplex detection was demonstrated for the human EGFR and KRAS genes using Eprobes with two different dyes. Combining amplification and melting curve analysis in a single-tube reaction provides powerful means for new mutation detection assays. Functioning as “sequence-specific dyes”, Eprobes hold great promises for future applications not only in PCR but also as hybridization probes in other applications.

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“…The major advantage of qPCR is its quantification of the target DNA sequence. Another significant improvement by qPCR is the increased speed of analysis, mainly due to the omission of the post-PCR detection step such as gelelectrophoresis (Mackay et al, 2004). Also, the fluorescence-sensitive equipment permits earlier amplicon detection and shortens the amplification cycle numbers required.…”

Section: Generamentioning

confidence: 99%

“…QPCR methods include the SYBR green assays and the Taq nuclease assays. Fluorescence will be generated from the binding of dye to double-stranded DNA (SYBR green) or from hydrolysis of a TaqMan probe (Taq nuclease assay), in which the TaqMan probe is considered to be more specific (Sivonen, 2008 (Mackay et al, 2004). However, qPCR has disadvantages involving the detection of fluorogenic chemistries and it also restricts multiplex amplifications (Mackay et al, 2004).…”

Section: Generamentioning

confidence: 99%

Dynamics of cyanobacteria and cyanotoxins in Kranji Reservoir

Te¹

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My PhD experience is one of the most precious learning times in my life. I have learnt a great deal from those who have worked with me over the years and gratefully acknowledge my debt to them. In the first place, most thanks should go to my supervisor Associate Professor Karina Yew-Hoong Gin. I would not have accomplished this thesis without her continuous support and encouragement, professional guidance and excellent advice during the whole study. Her passion for the research always inspires me. Through her, I learn to find the research the direction and appreciate the ups and downs of research life. She allowed me to work with a great flexibility but at the same time, she was never short on providing critical views and suggestions from different perspectives. She builds up good rapports with all her students, so when talk about her many PhD students and researchers always envy me for having such a wonderful supervisor. I would also like to express sincere gratitude to my second supervisor, Associate Professor Lim Teik Thye, for being exceptionally kind and helpful in assisting me to finish the study. My sincere thanks to my group members and previous colleagues and labmates including Dr Aw Tiong Gim for his generous assistance in laboratorial skills, research discussions and comments on the study and paper writings; Ms Uttara Ramaswamy who helped me in culturing techniques, and water samplings; Ms Yeo Been Hui for sharing same research interest, tedious field works and water quality analyses;

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Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

Cited by 9 publications

References 461 publications

Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

Dynamics of cyanobacteria and cyanotoxins in Kranji Reservoir

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