| 1. |
- Andersson, Daniel, 1979, et al.
(författare)
-
Properties of targeted preamplification in DNA and cDNA quantification
- 2015
-
Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1473-7159 .- 1744-8352. ; 15:8, s. 1085-1100
-
Forskningsöversikt (refereegranskat)abstract
- Objective: Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). Methods: To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. Result: The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. Conclusion: On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.
|
|
| 2. |
- Borrebaeck, Carl
(författare)
-
Advanced technologies and diagnostic spin-outs: an interview with Carl Borrebaeck.
- 2015
-
Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1744-8352 .- 1473-7159. ; 15:11, s. 1409-1410
-
Tidskriftsartikel (refereegranskat)abstract
- Interview with Professor Carl Borrebaeck DSc by Claire Raison (Commissioning Editor) Professor Carl Borrebaeck DSc, is the Director of CREATE Health (Lund, Sweden), a translational cancer center, previous Vice President of Lund University and specializes in immunotechnology, diagnostics and treatments for cancer. He has had remarkable success in co-founding collaborative working groups and related spin-out companies based in Lund. Professor Borrebaeck also serves as a member of the editorial advisory board for Expert Review of Molecular Diagnostics. Here, Professor Borrebaeck talks to Expert Review of Molecular Diagnostics about his experience and what is next for cutting-edge technology in cancer diagnostics.
|
|
| 3. |
- Donà, Valentina, et al.
(författare)
-
Recent advances in the development and use of molecular tests to predict antimicrobial resistance in Neisseria gonorrhoeae
- 2017
-
Ingår i: Expert Review of Molecular Diagnostics. - : Taylor & Francis Group. - 1473-7159 .- 1744-8352. ; 17:9, s. 845-859
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: The number of genetic tests, mostly real-time PCRs, to detect antimicrobial resistance (AMR) determinants and predict AMR in Neisseria gonorrhoeae is increasing. Several of these assays are promising, but there are important shortcomings and few assays have been adequately validated and quality assured.Areas covered: Recent advances, focusing on publications since 2012, in the development and use of molecular tests to predict gonococcal AMR for surveillance and for clinical use, advantages and disadvantages of these tests and of molecular AMR prediction compared with phenotypic AMR testing, and future perspectives for effective use of molecular AMR tests for different purposes.Expert commentary: Several challenges for direct testing of clinical, especially extra-genital, specimens remain. The choice of molecular assay needs to consider the assay target, quality controls, sample types, limitations intrinsic to molecular technologies, and specific to the chosen methodology, and the intended use of the test. Improved molecular- and particularly genome-sequencing-based methods will supplement AMR testing for surveillance purposes, and translate into point-of-care tests that will lead to personalized treatments, while sparing the last available empiric treatment option (ceftriaxone). However, genetic AMR prediction will never completely replace phenotypic AMR testing, which detects also AMR due to unknown AMR determinants.
|
|
| 4. |
- Jevtusevskaja, Jekaterina, et al.
(författare)
-
The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification
- 2017
-
Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1473-7159 .- 1744-8352. ; 17:4, s. 403-410
-
Forskningsöversikt (refereegranskat)abstract
- Background: The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assaysMethods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplificationResults: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases.Conclusion: These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.
|
|
| 5. |
- Krõlov, Katrin, et al.
(författare)
-
Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings
- 2017
-
Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1473-7159 .- 1744-8352. ; 17:12, s. 1117-1125
-
Tidskriftsartikel (refereegranskat)abstract
- Background: A variety of sample preparation techniques are used prior to nucleic acid amplification. However, their efficiency is not always sufficient and nucleic acid purification remains the preferred method for template preparation. Purification is difficult and costly to apply in point-of-care (POC) settings and there is a strong need for more robust, rapid, and efficient biological sample preparation techniques in molecular diagnostics. Methods: Here, the authors applied antimicrobial peptides (AMPs) for urine sample preparation prior to isothermal loop-mediated amplification (LAMP). AMPs bind to many microorganisms such as bacteria, fungi, protozoa and viruses causing disruption of their membrane integrity and facilitate nucleic acid release. Results: The authors show that incubation of E. coli with antimicrobial peptide cecropin P1 for 5 min had a significant effect on the availability of template DNA compared with untreated or even heat treated samples resulting in up to six times increase of the amplification efficiency. Conclusion: These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.
|
|
| 6. |
- Lindskog, Cecilia, et al.
(författare)
-
Immunohistochemistry-based prognostic biomarkers in NSCLC : novel findings on the road to clinical use?
- 2015
-
Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1473-7159 .- 1744-8352. ; 15:4, s. 471-490
-
Forskningsöversikt (refereegranskat)abstract
- Prognostication of non-small cell lung cancer is principally based on stage, age and performance status. This review provides an overview of 342 potential prognostic biomarkers in non-small cell lung cancer described between January 2008 and June 2013, evaluating the association between immunohistochemical protein expression and survival endpoint. Numerous studies proposed prognostic biomarkers, but many were only evaluated in a single patient cohort, and a large number of biomarkers revealed inconclusive findings when analyzed in more than one study. Only 26 proteins first described after 2008 (ALDH1A1, ANXA1, BCAR1, CLDN1, EIF4E, EZH2, FOLR1, FOXM1, IL7R, IL12RB2, KIAA1524, CRMP1, LOX, MCM7, MTA1, MTDH, NCOA3, NDRG2, NEDD9, NES, PBK, PPM1D, SIRT1, SLC7A5, SQSTM1 and WNT1) demonstrated a consistent prognostic association in two or more independent patient cohorts, thus qualifying as promising candidates for diagnostic use. Raised quality standards for study design and antibody validation, and integration of preclinical findings with clinical needs are clearly warranted.
|
|
| 7. |
- Ludvigsson, Johnny
(författare)
-
The clinical potential of low-level C-peptide secretion
- 2016
-
Ingår i: Expert Review of Molecular Diagnostics. - : TAYLOR & FRANCIS LTD. - 1473-7159 .- 1744-8352. ; 16:9, s. 933-940
-
Forskningsöversikt (refereegranskat)abstract
- Introduction: In all forms of diabetes, especially so in Type 1 diabetes (T1D), beta cell function is crucial for the course. The interest for residual beta cell function has been meagre, but increased in recent years with interventions to preserve function.Areas covered: Importance and clinical use of C-peptide in Type 1 diabetes. Data for this review were identified by searches of PubMed, and references from relevant articles using the search terms: type 1 diabetes, C-peptide, beta cell function, HbA1c, quality of life, complications. Abstracts and reports from meetings were not included.Expert commentary: C-peptide may help to get a correct diagnosis, and it is of practical clinical value to know degree of residual insulin secretion to diminish the risk of severe hypoglycaemia and ketoacidosis. Evidence shows that even a quite reduced beta cell function may play an important role for quality of life, for metabolic balance/control, possibility to avoid complications and even for long-term survival. Furthermore, the evidence is increasing for C-peptide being a hormone per se.
|
|
| 8. |
|
|
| 9. |
- Yilmaz, Aylin, 1974, et al.
(författare)
-
Neurofilament light chain protein as a marker of neuronal injury: review of its use in HIV-1 infection and reference values for HIV-negative controls
- 2017
-
Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1473-7159 .- 1744-8352. ; 17:8, s. 761-770
-
Forskningsöversikt (refereegranskat)abstract
- Introduction: Several CSF biomarkers of neuronal injury have been studied in people living with HIV. At this time, the most useful is the light subunit of the neurofilament protein (NFL). This major structural component of myelinated axons is essential to maintain axonal caliber and to facilitate effective nerve conduction. CSF concentrations of NFL provide a sensitive marker of CNS injury in a number of neurological diseases, including HIV-related neuronal injury. Areas Covered: In this review, the authors describe CSF NFL concentrations across the spectrum of HIV-infection, from its early acute phase to severe immunosuppression, with and without neurological conditions, and with and without antiretroviral treatment (n = 516). Furthermore, in order to provide more precise estimates of age-related upper limits of CSF NFL concentrations, the authors present data from a large number (n = 359) of HIV-negative controls. Expert Commentary: Recently a new ultrasensitive diagnostic assay for quantification of NFL in plasma has been developed, providing a convenient way to assess neuronal damage without having to perform a lumbar puncture. This review also considers our current knowledge of plasma NFL in HIV CNS infection.
|
|
