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A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR.

ABSTRACT: Non-specific tail sequences are often added to the 5'-terminus of primers to improve the robustness and overall performance of diagnostic assays. Despite the widespread use of tailed primers, the underlying working mechanism is not well understood. To address this problem, we conducted a detailed in vitro and in silico analysis of the enhancing effect of primer tailing on 2 well-established foot-and-mouth disease virus (FMDV) RT-qPCR assays using an FMDV reference panel. Tailing of the panFMDV-5UTR primers mainly affected the shape of the amplification curves. Modelling of the raw fluorescence data suggested a reduction of the amplification efficiency due to the accumulation of inhibitors. In depth analysis of PCR products indeed revealed the rapid accumulation of forward-primer derived artefacts. More importantly, tailing of the forward primer delayed artefacts formation and concomitantly restored the sigmoidal shape of the amplification curves. Our analysis also showed that primer tailing can alter utilisation patterns of degenerate primers and increase the number of primer variants that are able to participate in the reaction. The impact of tailed primers was less pronounced in the panFMDV-3D assay with only 5 out of 50 isolates showing a clear shift in Cq values. Sequence analysis of the target region of these 5 isolates revealed several mutations in the inter-primer region that extend an existing hairpin structure immediately downstream of the forward primer binding site. Stabilisation of the forward primer with either a tail sequence or cationic spermine units restored the sensitivity of the assay, which suggests that the enhancing effect in the panFMDV-3D assay is due to a more efficient extension of the forward primer. ur results show that primer tailing can alter amplification through various mechanisms that are determined by both the assay and target region. These findings expand our understanding of primer tailing and should enable a more targeted and efficient use of tailed primers.

SUBMITTER: Vandenbussche F

PROVIDER: S-EPMC5056738 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR.

Vandenbussche Frank F Mathijs Elisabeth E Lefebvre David D De Clercq Kris K Van Borm Steven S

PloS one 20161010 10

Non-specific tail sequences are often added to the 5'-terminus of primers to improve the robustness and overall performance of diagnostic assays. Despite the widespread use of tailed primers, the underlying working mechanism is not well understood. To address this problem, we conducted a detailed in vitro and in silico analysis of the enhancing effect of primer tailing on 2 well-established foot-and-mouth disease virus (FMDV) RT-qPCR assays using an FMDV reference panel. Tailing of the panFMDV-5 ...[more]

PMID: 27723800

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Project description:BACKGROUND: Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora. RESULTS: Design of specific primers and probe was achieved based on comparison of 108 published bifidobacterial 16S rDNA sequences with the recently published sequence of the human faecal isolate B. thermophilum RBL67. Specificity of the primer was tested in silico by similarity search against the sequence database and confirmed experimentally by PCR amplification on 17 Bifidobacterium strains, representing 12 different species, and two Lactobacillus strains. The qPCR assay developed was linear for B. thermophilum RBL67 DNA quantities ranging from 0.02 ng/microl to 200 ng/microl and showed a detection limit of 10(5) cells per gram faeces. The application of this new qPCR assay allowed to detect the presence of B. thermophilum in one sample from a 6-month old breast-fed baby among 17 human faecal samples tested. Additionally, the specific qPCR primers in combination with selective plating experiments led to the isolation of F9K9, a faecal isolate from a 4-month old breast-fed baby. The 16S rDNA sequence of this isolate is 99.93% similar to that of B. thermophilum RBL67 and confirmed the applicability of the new qPCR assay in faecal samples. CONCLUSION: A new B. thermophilum-specific qPCR assay was developed based on species-specific target nucleotides in the 16S rDNA. It can be used to further characterize the composition of the bifidobacterial community in the human gastrointestinal tract. Until recently, B. thermophilum was considered as a species of animal origin, but here we confirm with the application of this new PCR assay the presence of B. thermophilum strains in the human gut.

Project description:Ascidians are invertebrate chordates, with swimming chordate tadpole larvae that have distinct heads and tails. The head contains the small brain, sensory organs, including the ocellus (light) and otolith (gravity) and the presumptive endoderm, while the tail has a notochord surrounded by muscle cells and a dorsal nerve cord. One of the chordate features is a post-anal tail. Ascidian tadpoles are nonfeeding, and their tails are critical for larval locomotion. After hatching the larvae swim up toward light and are carried by the tide and ocean currents. When competent to settle, ascidian tadpole larvae swim down, away from light, to settle and metamorphose into a sessile adult. Tunicates are classified as chordates because of their chordate tadpole larvae; in contrast, the sessile adult has a U-shaped gut and very derived body plan, looking nothing like a chordate. There is one group of ascidians, the Molgulidae, where many species are known to have tailless larvae. The Swalla Lab has been studying the evolution of tailless ascidian larvae in this clade for over 30 years and has shown that tailless larvae have evolved independently several times in this clade. Comparison of the genomes of two closely related species, the tailed Molgula oculata and tailless Molgula occulta reveals much synteny, but there have been multiple insertions and deletions that have disrupted larval genes in the tailless species. Genomics and transcriptomics have previously shown that there are pseudogenes expressed in the tailless embryos, suggesting that the partial rescue of tailed features in their hybrid larvae is due to the expression of intact genes from the tailed parent. Yet surprisingly, we find that the notochord gene regulatory network is mostly intact in the tailless M. occulta, although the notochord does not converge and extend and remains as an aggregate of cells we call the "notoball." We expect that eventually many of the larval gene networks will become evolutionarily lost in tailless ascidians and the larval body plan abandoned, with eggs developing directly into an adult. Here we review the current evolutionary and developmental evidence on how the molgulids lost their tails.

Dataset Information

A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR.

Publications

A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR.

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