Duplicate reactions were run using fold RNA dilutions ng to 0. Reactions were run in triplicate using fold dilutions of in vitro transcript 10 7 down to 10 copies. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times.
Open lightbox. Reactions were run in triplicate. NTC: no template control. You are not authorized to download the resource. Sort options Sort alphabetically. What should I use as a standard for absolute quantification in real-time PCR? FAQ ID What is a QuantiTect Primer Assay?
Do you offer trial-kit sizes for the new QuantiFast Kits? How do you achieve fast cycling, yet still deliver the same performance in PCR as that achieved with standard cycling? How much time will be saved when switching from standard cycling to fast cycling with QuantiFast Kits?
QuantiTect Primer Assays are bioinformatically validated, genomewide primer sets. Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?
What is the threshold cycle or Ct value? Why do replicates in real-time PCR have different plateau heights? Why does my realtime PCR assay quality decrease over time? The mcr-1 can be detected as low as 1 copy per 1,, copies of 16s rRNA in the feces samples, and the highest abundance of mcr-1 could reach 10 5 copies per 1,, copies of 16s rRNA Figure 2.
Relative abundance was the most popular method to measure the abundance of antibiotic resistance genes in the feces and soil samples, as the composition and abundance of microbiome varies a lot among different feces and soil samples Bontron et al.
In this study, both mcr-1 and mcr-3 has been successfully detected in the cultured bacteria from natural animal and environment isolates. However, mcr-2 has not been validated in the cultured bacteria, as lacking mcr-2 carrying bacteria in our lab.
The specificity of the primers has been confirmed by both traditional PCR and melting curve analysis. The limitation of this method was all three mcr genes could not be detected in one reaction comparing with the Taqman assay. However, this method does have the flexibility for screening different mcr genes with any combination at relative lower cost, as the co-existence of mcr genes has not been reported in the clinical medicine, and it is not necessary to screen all three mcr genes.
Recently, only one case was reported about the co-occurrence of mcr-1 and mcr-3 in one E. Also considering the number of novel mcr gene has been growing, the flexible combination of the detection assay does have its advantage. In conclusion, this SYBR Green-based real-time PCR assay is a rapid, sensitive, and highly specific detection assay for the mcr-1, mcr-2, and mcr-3 genes either from cultured bacteria, feces, and soil samples.
It is easy to perform in any laboratory having at its disposal a qPCR machine. This rapid technique may be used for the evaluation of the prevalence of this resistance trait in humans and animals surveillance studies. In addition, it will be a valuable tool for fast screening and quantifying all mcr genes not only in the bacterial, but also in feces and soil samples.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Figure S1. Multiple sequence alignment of mcr-1 variants. Figure S2. A—C were the electrophoresis gel left and melting curve right of mcr-1, mcr-2 , and mcr M: Marker. NTC: negative control. Bontron, S. Real-time PCR for detection of plasmid-mediated polymyxin resistance mcr-1 from cultured bacteria and stools.
Hembach, N. Occurrence of the mcr-1 colistin resistance gene and other clinically relevant antibiotic resistance genes in microbial populations at different municipal wastewater treatment plants in germany.
Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September Euro Surveill. Huang, X. High prevalence of colistin resistance and mcr-1 gene in Escherichia coli isolated from food animals in china.
Liu, Y. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect. Similarly, an assay could be developed to identify cyclin D1 and MDM2 gene amplification. The former is a prognostic marker in breast carcinoma [ 29 ] while the latter plays a role in the pathogenesis of a variety of tumours [ 30 ]. To date such genetic abnormalities can only be detected unequivocally by FISH.
This requires live material and is therefore not always possible. This assay could be applied to any gene implicated in an autosomal dominant disease, in order to determine whether whole or partial gene deletion is a significant cause of disease. In these conditions, one copy of the tumour suppressor gene is either mutated or deleted in the germ line, while the second copy is lost or mutated in the tumour itself. Genetic counselling could therefore benefit from a quick and reliable assay to determine carriers of such deletions.
Two reference loci were also used: exon 19 of the cystic fibrosis transmembrane conductance regulator CFTR at 7q31 and ubiquitin-conjugating enzyme pseudogene UBE2L2 at 12q Moreover, experiments were performed in triplicate to ensure reproducibility of the technique. CAS Google Scholar. Embo J. Hum Genet.
Article Google Scholar. Cancer Res. Human Molecular Genetics. J Med Genet. Nature Genetics. Google Scholar. Cancer Research. Nature Immunology. British Society of Rheumatology. Edited by: Ligand Assay.
Ramachandra C, Melnick SJ: Multidrug resistance in human tumors : molecular diagnosis and clinical significance. Download references. You can also search for this author in PubMed Google Scholar. Correspondence to Frederique Ponchel. FP, conceived the study, designed it, performed it and supervised the real time PCR work of other authors. Reprints and Permissions. Ponchel, F. BMC Biotechnol 3, 18 Download citation.
Received : 16 April Accepted : 13 October Published : 13 October Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. Results We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay.
Conclusion Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.
Background The detection and quantification of gene rearrangement, amplification, translocation or deletion is a significant problem, both in research and in a clinical diagnostic setting.
Figure 1. Full size image. Figure 2. Table 1 Real-time nucleotide primer sequences Full size table. Figure 3. Figure 4. Figure 5. Figure 6. References 1. CAS Google Scholar 2. CAS Google Scholar 6. Article Google Scholar 9. CAS Google Scholar Article Google Scholar Google Scholar View author publications. Additional information Author's contribution FP, conceived the study, designed it, performed it and supervised the real time PCR work of other authors.
CT, performed the deletion work. FTL, performed the transgenic mouse work. SHD, provided technical assistance to FP. SMB, provided cell lines. CFI, supervised the deletion work. JDI, is head of group. AFM, is head of department. Rights and permissions Reprints and Permissions.
About this article Cite this article Ponchel, F. Copy to clipboard. Contact us Submission enquiries: bmcbiotechnology biomedcentral.
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