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.

Advances in liquid biopsy-based markers in NSCLC.

Lung cancer is the second most-frequently occurring cancer and the leading cause of cancer-associated deaths worldwide. Non-small cell lung cancer (NSCLC), the most common type of lung cancer is often diagnosed in middle or advanced stages and have poor prognosis. Diagnosis of disease at an early stage is a key factor for improving prognosis and reducing mortality, whereas, the currently used diagnostic tools are not sufficiently sensitive for early-stage NSCLC. The emergence of liquid biopsy has ushered in a new era of diagnosis and management of cancers, including NSCLC, since analysis of circulating tumor-derived components, such as cell-free DNA (cfDNA), circulating tumor cells (CTCs), cell-free RNAs (cfRNAs), exosomes, tumor-educated platelets (TEPs), proteins, and metabolites in blood or other biofluids can enable early cancer detection, treatment selection, therapy monitoring and prognosis assessment. There have been great advances in liquid biopsy of NSCLC in the past few years. Hence, this chapter introduces the latest advances on the clinical application of cfDNA, CTCs, cfRNAs and exosomes, with a particular focus on their application as early markers in the diagnosis, treatment and prognosis of NSCLC.

Rapid detection of EGFR mutation in CTCs based on a double spiral microfluidic chip and the real-time RPA method.

Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumors and spread into the peripheral bloodstream. Mutation detection in CTCs can reveal vital genetic information about the tumors and can be used for "liquid biopsy" to indicate cancer treatment and targeted medication. However, current methods to measure the mutations in CTCs are based on PCR or DNA sequencing which are cumbersome and time-consuming and require sophisticated equipment. These largely limited their applications especially in areas with poor healthcare infrastructure. Here we report a simple, convenient, and rapid method for mutation detection in CTCs, including an example of a deletion at exon 19 (Del19) of the epidermal growth factor receptor (EGFR). CTCs in the peripheral blood of NSCLC patients were first sorted by a double spiral microfluidic chip with high sorting efficiency and purity. The sorted cells were then lysed by proteinase K, and the E19del mutation was detected via real-time recombinase polymerase amplification (RPA). Combining the advantages of microfluidic sorting and real-time RPA, an accurate mutation determination was realized within 2 h without professional operation or complex data interpretation. The method detected as few as 3 cells and 1% target variants under a strongly interfering background, thus, indicating its great potential in the non-invasive diagnosis of E19del mutation for NSCLC patients. The method can be further extended by redesigning the primers and probes to detect other deletion mutations, insertion mutations, and fusion genes. It is expected to be a universal molecular diagnostic tool for real-time assessment of relevant mutations and precise adjustments in the care of oncology patients.

Centrifugal microfluidic-based multiplex recombinase polymerase amplification assay for rapid detection of SARS-CoV-2.

The COVID-19 pandemic has spread worldwide, and rapid detection of the SARS-CoV-2 virus is crucial for infection surveillance and epidemic control. This study developed a centrifugal microfluidics-based multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay for endpoint fluorescence detection of the E, N, and ORF1ab genes of SARS-CoV-2. The microscope slide-shaped microfluidic chip could simultaneously accomplish three target genes and one reference human gene (i.e., ACTB) RT-RPA reactions in 30 min, and the sensitivity was 40 RNA copies/reaction for the E gene, 20 RNA copies/reaction for the N gene, and 10 RNA copies/reaction for the ORF1ab gene. The chip demonstrated high specificity, reproducibility, and repeatability. Chip performance was also evaluated using real clinical samples. Thus, this rapid, accurate, on-site, and multiplexed nucleic acid test microfluidic chip would significantly contribute to detecting patients with COVID-19 in low-resource settings and point-of-care testing (POCT) and, in the future, could be used to detect emerging new variants of SARS-CoV-2.

A Resistance-Based Microfluidic Chip for Deterministic Single Cell Trapping Followed by Immunofluorescence Staining.

Microchips are fundamental tools for single-cell analysis. Although various microfluidic methods have been developed for single-cell trapping and analysis, most microchips cannot trap single cells deterministically for further analysis. In this paper, we describe a novel resistance-based microfluidic chip to implement deterministic single-cell trapping followed by immunofluorescence staining based on the least flow resistance principle. The design of a large circular structure before the constriction and the serpentine structure of the main channel made the flow resistance of the main channel higher than that of the trapping channel. Since cells preferred to follow paths with lower flow resistance, this design directed cells into the capture sites and improved single-cell trapping efficiency. We optimized the geometric parameters using numerical simulations. Experiments using A549 and K562 cell lines demonstrated the capability of our chip with (82.7 ± 2.4)% and (84 ± 3.3)% single-cell trapping efficiency, respectively. In addition, cells were immobilized at capture sites by applying the pulling forces at the outlet, which reduced the cell movement and loss and facilitated tracking of the cell in real time during the multistep immunofluorescence staining procedure. Due to the simple operation, high-efficiency single-cell trapping and lower cell loss, the proposed chip is expected to be a potential analytical platform for single tumor cell heterogeneity studies and clinical diagnosis.

Dynamic cerebrospinal fluid analyses of severe pseudorabies encephalitis.

We reported a severe human pseudorabies encephalitis case and described a dynamic clinical manifestation with cerebrospinal fluid analyses and cytological and serological evaluation, which may elucidate the mechanism of PRV infection and facilitate clinical diagnosis and treatment in human.

[Correlation of coagulation indicators with inflammatory markers for sepsis in the patients with hematological malignancies].

This study was aimed to investigate the correlation of coagulation indicators [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB), antithrombinIII (ATIII), D-dimer (D-D) levels] with inflammatory markers [procalcitonin (PCT), C reactive protein (CRP), interleukin-6 (IL-6), serum amyloid A (SAA)] for sepsis in hematologic malignancy patients. A total of 326 febrile in patients with hematologic diseases from 2062 patients in West China Hospital, Sichuan University from March 2011 to April 2013 were retrospectively analyzed. The patients were divided into sepsis group(n = 72), non-sepsis group(n = 176) and non-sepsis with low Alb group (n = 78) according to blood culture. The results showed that the values of PT, APTT, D-dimer, Plt in sepsis group were higher than those in non-sepsis group, and the difference between them was statistically significant. While the ATIII level in the sepsis group was lower than that in non-sepsis group, and the difference between them was statistically significant (P < 0.05). And the four inflammatory biomarkers in the sepsis patients were higher than those in non-sepsis patients (P < 0.05). TT and FIB level were not significantly different (P > 0.05). There was not a significant difference in these indicators between non-sepsis group and non-sepsis with low Alb group. The correlation analysis suggested that the level of PCT positively correlated with APTT, D-dimer level (P < 0.05); and negatively correlated with the ATIII (P < 0.05). It is concluded that sepsis results in the concurrent activation of inflammatory and procoagulant pathways. The hematologic malignancy patients with sepsis have an obviously higher systemic inflammatory response, and accompanied with coagulation dysfunction.