Urine cell-free DNA as a promising biomarker for early detection of non-small cell lung cancer.

BACKGROUND: While blood-derived cell-free DNA has been shown to be a candidate biomarker able to provide diagnostic and prognostic insight in cancer patients, little is known regarding the potential application of urine cell-free DNA (ucfDNA) in diagnosis of cancer. Thus, the aim of this study was to investigate ucfDNA concentration and integrity index as potential biomarkers for early detection of non-small-cell lung cancer (NSCLC). METHODS: Urine samples were collected from 35 healthy controls and 55 NSCLC patients at various tumor node metastasis (TNM) stages. Two long interspersed nuclear element 1 (LINE1) fragments (LINE1-97 and 266 bp) were quantified via quantitative real-time PCR (qPCR). DNA integrity index was calculated as the ratio of LINE1-266/LINE-97. RESULTS: LINE1 fragments concentrations of ucfDNA (LINE1-97, 266 bp) were significantly higher in NSCLC patients with stage III/IV than in stage I/II and in healthy controls. The receiver operating characteristic (ROC) curves for discriminating patients with stage III/IV from healthy controls had areas under the curves (AUC) of 0.84 and 0.886, respectively. Moreover, ucfDNA integrity LINE1-266/97 was significantly higher in patients with stage III/IV than in stage I/II and in healthy controls. The AUC of ROC curve for discriminating patients with stage III/IV from healthy controls was 0.800. Furthermore, LINE1-266 fragment concentration was significantly higher in lymph node metastasis (LNM)-positive patients relative to LNM-negative patients. The ROC curve for discriminating LNM-positive from LNM-negative patients had an AUC of 0.822. CONCLUSION: UcfDNA could serve as a promising biomarker for early detection of NSCLC.

Development of a novel integrated isothermal amplification system for detection of bacteria-spiked blood samples.

Bloodstream infection (BSI) caused by bacteria is highly pathogenic and lethal, and easily develops whole-body inflammatory state. Immediate identification of disease-causing bacteria can improve patient prognosis. Traditional testing methods are not only time-consuming, but such tests are limited to laboratories. Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) holds great promise for rapid nucleic acid detection, but the uncapping operation after amplification easily contaminates laboratories. Therefore, the establishment of a more effective integrated isothermal amplification system has become an urgent problem to be solved. In this study, we designed and fabricated a hermetically sealed integrated isothermal amplification system. Combining with this system, a set of RPA-LFD assays for detecting S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI were established and evaluated. The whole process could be completed in less than 15 min and the results can be visualized by the naked eye. The developed RPA-LFD assays displayed a good sensitivity, and no cross-reactivity was observed in seven similar bacterial genera. The results obtained with 60 clinical samples indicated that the developed RPA-LFD assays had high specifcity and sensitivity for identifying S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI. In conclusion, our results showed that the developed RPA-LFD assay is an alternative to existing PCR-based methods for detection of S. aureus, K. peneumoniae, P. aeruginosa, and H. influenza in BSI in primary hospitals.

Enhanced Separation Efficiency and Purity of Circulating Tumor Cells Based on the Combined Effects of Double Sheath Fluids and Inertial Focusing.

Circulating tumor cells (CTCs) play a crucial role in solid tumor metastasis, but obtaining high purity and viability CTCs is a challenging task due to their rarity. Although various works using spiral microchannels to isolate CTCs have been reported, the sorting purity of CTCs has not been significantly improved. Herein, we developed a novel double spiral microchannel for efficient separation and enrichment of intact and high-purity CTCs based on the combined effects of two-stage inertial focusing and particle deflection. Particle deflection relies on the second sheath to produce a deflection of the focused sample flow segment at the end of the first-stage microchannel, allowing larger particles to remain focused and entered the second-stage microchannel while smaller particles moved into the first waste channel. The deflection of the focused sample flow segment was visualized. Testing by a binary mixture of 10.4 and 16.5 µm fluorescent microspheres, it showed 16.5 µm with separation efficiency of 98% and purity of 90% under the second sheath flow rate of 700 µl min-1. In biological experiments, the average purity of spiked CTCs was 74% at a high throughput of 1.5 × 108 cells min-1, and the recovery was more than 91%. Compared to the control group, the viability of separated cells was 99%. Finally, we validated the performance of the double spiral microchannel using clinical cancer blood samples. CTCs with a concentration of 2-28 counts ml-1 were separated from all 12 patients' peripheral blood. Thus, our device could be a robust and label-free liquid biopsy platform in inertial microfluidics for successful application in clinical trials.

Preliminary Evaluation of Rapid Visual Identification of Burkholderia pseudomallei Using a Newly Developed Lateral Flow Strip-Based Recombinase Polymerase Amplification (LF-RPA) System.

Burkholderia pseudomallei is an important infectious disease pathogen that can cause melioidosis. Melioidosis is mainly prevalent in Thailand, northern Australia and southern China and has become a global public health problem. Early identification of B. pseudomallei is of great significance for the diagnosis and prognosis of melioidosis. In this study, a simple and visual device combined with lateral flow strip-based recombinase polymerase amplification (LF-RPA) was developed, and the utility of the LF-RPA assay for identifying B. pseudomallei was evaluated. In order to screen out the optimal primer probe, a total of 16 pairs of specific primers targeting the orf2 gene of B. pseudomallei type III secretion system (T3SS) cluster genes were designed for screening, and F1/R3 was selected as an optimal set of primers for the identification of B. pseudomallei, and parameters for LF-RPA were optimized. The LF-RPA can be amplified at 30-45°C and complete the entire reaction in 5-30 min. This reaction does not cross-amplify the DNA of other non-B. pseudomallei species. The limit of detection (LOD) of this assay for B. pseudomallei genomic DNA was as low as 30 femtograms (fg), which was comparable to the results of real-time PCR. Moreover, 21 clinical B. pseudomallei isolates identified by 16S rRNA gene sequencing were retrospectively confirmed by the newly developed LF-RPA system. Our results showed that the newly developed LF-RPA system has a simple and short time of operation and has good application prospect in the identification of B. pseudomallei.