Comparison of droplet digital PCR and conventional quantitative PCR for measuring EGFR gene mutation.

Early detection of epidermal growth factor receptor (EGFR) mutation, particularly EGFR T790M mutation, is of clinical significance. The aim of the present study was to compare the performances of amplification refractory mutation system-based quantitative polymerase chain reaction (ARMS-qPCR) and droplet digital polymerase chain reaction (ddPCR) approaches in the detection of EGFR mutation and explore the feasibility of using ddPCR in the detection of samples with low mutation rates. EGFR gene mutations in plasmid samples with different T790M mutation rates (0.1-5%) and 10 clinical samples were detected using the ARMS-qPCR and ddPCR approaches. The results demonstrated that the ARMS-qPCR method stably detected the plasmid samples (6,000 copies) with 5 and 1% mutation rates, while the ddPCR approach reliably detected those with 5% (398 copies), 1% (57 copies), 0.5% (24 copies) and 0.1% (average 6 copies) mutation rates. For the 10 clinical samples, the results for nine samples by the ARMS-qPCR and ddPCR methods were consistent; however, the sample N006, indicated to be EGFR wild-type by ARMS-qPCR, was revealed to have a clear EGFR T790M mutation with seven copies of mutant alleles in a background of 6,000 wild-type copies using ddPCR technology. This study demonstrates the feasibility of applying the ddPCR system to detect EGFR mutation and identified the advantage of ddPCR in the detection of samples with a low EGFR mutation abundance, particularly the secondary EGFR T790M resistance mutation, which enables early diagnosis before acquired resistance to tyrosine kinase inhibitors becomes clinically detectable.

Evaluation and identification of microRNA-106 in the diagnosis of cancer: a meta-analysis.

Recently, extensive research has identified the non-invasive and cost-effective biomarker microRNA-106 (miR-106) in cancer detection. However, inconsistent results have prevented its usage in clinical. Therefore, we conducted this meta-analysis aimed to systematically determine diagnostic accuracy of miR-106 in distinguishing patients with cancer from cancer-free controls and further evaluate its value serving as a biomarker in clinical. We conducted a systematically literature search in databases (PubMed, web of science, Embase and the Cochrane Library) collecting relevant articles up to July 22th, 2014. The overall diagnostic accuracy of miR-106 was assessed by the following indexes: sensitivity, specificity, PLR, NLR and DOR. The SROC curve with AUC value was also generated for the assessment. Due to the significant heterogeneity, the random effects approach was chosen in our analysis and meta-regression was performed to explore the potential source of it. We also tested potential presence of publication bias using Deeks' funnel plots test. Stata 12.0 statistical software was used for analysis in the present study. Overall, the 11 studies involving 756 cancer patients and 834 controls were considered eligible in our analysis. The results in our work showed that sensitivity of 0.57 (95% CI: 0.44-0.68) and specificity of 0.85 (95% CI: 0.72-0.92), with the under area AUC value of 0.75 (95% CI: 0.71-0.79) for miR-106 assay. Additionally, the combined PLR, NLR and DOR describing the discriminatory ability were 3.7 (95% CI: 2.2-6.2), 0.51 (95% CI: 0.42-0.62) and 7 (95% CI: 4-12) in the present analysis. The results in our meta-analysis showed that miR-106 had moderate accuracy in identifying cancer patients. Thus, further larger-scale prospective studies are needed to improve the diagnostic efficiency and explore the combination of miR-106 and other biomarkers with more pronounced accuracy.

Simultaneous determination of esculin and its metabolite esculetin in rat plasma by LC-ESI-MS/MS and its application in pharmacokinetic study.

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of esculin and its metabolite esculetin in rat plasma. After addition of internal standards scopoletin, the plasma sample was pretreated by solid-phase extraction (SPE), and separated on a reversed phase C(18) column with a mobile phase of 0.01% formic acid in water (solvent A) and methanol (solvent B) using isocratic elution (A:B=20:80, v/v). The detection of target compounds was done in multiple reaction monitoring (MRM) mode. The MRM detection was operated in the negative ESI mode using the transitions of m/z 339.1 ([M-H](-))→176.7 for esculetin, m/z 176.9 ([M-H](-))→133.0 and m/z 191.0 ([M-H](-))→175.9 for scopoletin. The standard curves, which ranged from 25 to 3200 ng/mL for esculin with the lowest limit of quantification (LLOQ) of 0.25 ng/mL and from 1.25 to 160 ng/mL for esculetin with the LLOQ of 1.25 ng/mL, were fitted to a 1/x weighted quadratic regression model. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD<8.73%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to study the pharmacokinetics of esculin and its metabolite esculetin in rat plasma after oral administration of esculin at a dose of 100mg/kg.

[Hydrogen production and enzyme activity of acidophilic strain X-29 at different C/N ratio].

Some fermentative bacteria can produce hydrogen by utilizing carbohydrate and other kinds of organic compounds as substrates. Hydrogen production was also determined by both the limiting of growth and related enzyme activity in energy metabolism. Carbon and nitrogen are needed for the growth and metabolism of microorganisms. In addition, the carbon/nitrogen (C/N) ratio can influence the material metabolized and the energy produced. In order to improve the hydrogen production efficiency of the bacteria, we analyzed the effect of different C/N ratios on hydrogen production and the related enzyme activities in the acidophilic strain X-29 using batch test. The results indicate that the differences in the metabolism level and enzyme activity are obvious at different C/N ratios. Although the difference in liquid fermentative products produced per unit of biomass is not obvious, hydrogen production is enhanced at a specifically determined ratio. At a C/N ratio of 14 the accumulative hydrogen yield of strain X-29 reaches the maximum, 2210.9 mL/g. At different C/N ratios, the expression of hydrogenase activity vary; the activity of hydrogenase decrease quickly after reaching a maximum along with the fermentation process, but the time of expression is short. The activity of alcohol dehydrogenase (ADH) tend to stabilize after reaching a peak along with the fermentation process, the difference in expression activity is little, and the expression period is long at different C/N ratios. At a C/N ratio of 14 hydrogenase and ADH reach the maximum 2.88 micromol x (min x mg)(-1) and 33.2 micromol x (min x mg)(-1), respectively. It is shown that the C/N ratio has an important effect on enhancing hydrogen production and enzyme activity.