Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation

Rajagopal, Aditya; Yurk, Dominic; Shin, Claudia; Menge, Karen; Jacky, Lucien; Fraser, Scott E.; Tombrello, T. A.; Tsongalis, Gregory J.

doi:10.1038/s41598-018-37732-y

Cited by 29 publications

(31 citation statements)

References 18 publications

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“…This primer combination was used on selected genomic DNAs from M. avium subspecies. The single DNA amplification was able to distinguish each subspecies on the basis of size by gel electrophoresis ( Figure 3 ), but can also be adapted to real time PCR using different Taqman probes and concentrations ( 32 ).…”

Section: Resultsmentioning

confidence: 99%

Diagnostic Sequences That Distinguish M. avium Subspecies Strains

et al. 2021

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Over a decade ago Mycobacterium avium subspecies paratuberculosis (Map) specific genes were initially identified in a whole genome context by comparing draft genome sequences of Map strain K-10 with Mycobacterium avium subspecies hominissuis (Mah) strain 104. This resulted in identification of 32 Map specific genes, not including repetitive elements, based on the two-genome comparison. The goal of this study was to define a more complete catalog of M. avium subspecies-specific genes. This is important for obtaining additional diagnostic targets for Johne's disease detection and for understanding the unique biology, evolution and niche adaptation of these organisms. There are now over 28 complete genome sequences representing three M. avium subspecies, including avium (Maa), Mah, and Map. We have conducted a comprehensive comparison of these genomes using two independent pan genomic comparison tools, PanOCT and Roary. This has led to the identification of more than 250 subspecies defining genes common to both analyses. The majority of these genes are arranged in clusters called genomic islands. We further reduced the number of diagnostic targets by excluding sequences having high BLAST similarity to other mycobacterial species recently added to the National Center for Biotechnology Information database. Genes identified as diagnostic following these bioinformatic approaches were further tested by DNA amplification PCR on an additional 20 M. avium subspecies strains. This combined approach confirmed 86 genes as Map-specific, seven as Maa-specific and three as Mah-specific. A single-tube PCR reaction was conducted as a proof of concept method to quickly distinguish M. avium subspecies strains. With these novel data, researchers can classify isolates in their freezers, quickly characterize clinical samples, and functionally analyze these unique genes.

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Section: Resultsmentioning

confidence: 99%

Diagnostic Sequences That Distinguish M. avium Subspecies Strains

et al. 2021

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“…We are also the first to demonstrate multiplex RT-PCR of single cells in nanoliter droplets, greatly enhancing the capability of our platform to perform gene expression profiling of single cells and identify highly specific populations of cells. Our assay can potentially be engineered to detect more genes via further optimization and approaches such as amplitude modulation, where the relative TaqMan probe concentrations amongst targets are varied to yield distinctive PCR curves 83,84 . Since PCR primers and probes can be designed for any DNA and RNA targets, this technique can be readily adapted to analyze any cell population of interest.…”

Section: Discussionmentioning

confidence: 99%

Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection

Tran

Wan

et al. 2021

Sci Rep

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Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.

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“…This enables compatibility of the TBP assay regents with existing four- or five-channel real-time PCR instruments that are found in most clinical laboratories. The ABI 7500 Fast DX instrument was used for RT-PCR in our study; however, previous studies have demonstrated equivalent performance between ABI PCR systems and the LightCycler 480 system (Roche) (15). Thus, we find the HDPCR Tickborne Panel provides a rapid multiplexed molecular approach to identify nine pathogens or pathogen groups commonly associated with tick-borne illness in the United States and can serve as a viable adjunct for the laboratory diagnosis of tick-borne infections.…”

Section: Discussionmentioning

confidence: 99%

“…A mathematical algorithm is used to analyze raw RT-PCR amplification data (PCR curves) to identify and differentiate up to six unique targets per real-time PCR fluorometric channel. This HDPCR approach has previously been applied to successfully identify nine viral agents in simulated nasopharyngeal specimens across a wide concentration of 10 1 to 10 5 genomic copies/PCR (15). Single channel multiplexing allows expansion of multiplex capabilities enabling identification of the nine TBP assay targets and an internal control using just four channels of a standard real-time PCR instrument (Fig.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of a Novel Multiplex High-Definition PCR Assay for Detection of Tick-Borne Pathogens in Whole-Blood Specimens

et al. 2019

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The prevalence of tick-borne infections has been steadily increasing in both number and geographic distribution in the United States and abroad. This increase, in conjunction with the continued recognition of novel pathogens transmitted by ticks, has made accurate diagnosis of these infections challenging. Mainstay serologic tests are insensitive during the acute phase of infection and are often cross-reactive with similar pathogenic and nonpathogenic organisms.

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Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation

Cited by 29 publications

References 18 publications

Diagnostic Sequences That Distinguish M. avium Subspecies Strains

Diagnostic Sequences That Distinguish M. avium Subspecies Strains

Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection

Evaluation of a Novel Multiplex High-Definition PCR Assay for Detection of Tick-Borne Pathogens in Whole-Blood Specimens

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