Loop-mediated isothermal amplification using self-avoiding molecular recognition systems and antarctic thermal sensitive uracil-DNA-glycosylase for detection of nucleic acid with prevention of carryover contamination.

Loop-mediated isothermal amplification (LAMP) is the most popular technique to amplify nucleic acid sequence without the use of temperature cycling. However, LAMP is often confounded by false-positive results, arising from interactions between (hetero-dimer) or within (self-dimerization) primers, off-target hybrids and carryover contaminants. Here, we devised a new LAMP technique that is self-avoiding molecular recognition system (SAMRS) components and antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) enzyme-assisted, termed AUDG-SAMRS-LAMP. Incorporating SAMRS components into 3'-ends of LAMP primers can improve assay's specificity, which completely prevents the non-specific amplification yielding from off-target hybrids and undesired interactions between or within primers. Adding AUDG into reaction mixtures can effectively eliminate the false-positive results arising from carryover contamination, thus the genuine positive reactions are generated from the amplification of target templates. Furthermore, AUDG-SAMRS-LAMP results are confirmed using a new analysis strategy, which is developed for detecting LAMP amplicons by lateral flow biosensor (LFB). Only a single labeled primer is required in the analysis system, thus the false positive results arising from hybridization (the labeled primer and probe, or between two labeled primers) are avoided. Hence, the SAMRS components, AUDG and LFB convert traditional LAMP from a technique suited for the research laboratory into one that has practical value in the field of diagnosis. Human Tuberculosis (TB) is caused by infection with members of Mycobacterium tuberculosis complex (MTC), which are detected by the AUDG-SAMRS-LAMP technique to demonstrate the availability of target analysis. The proof-of-concept method can be reconfigured to detect various nucleic acids by redesigning the specific primers.

Expression of EphrinB2 and EphB4 in glioma tissues correlated to the progression of glioma and the prognosis of glioblastoma patients.

OBJECTIVE: The ligand EphrinB2 and the corresponding receptor EphB4 are up-regulated and involved in tumour growth in various human cancers. However, little is known about how this receptor-ligand complex contributes to the progression of glioma. This prompted us to study the association between the expressions of EphrinB2 and EphB4, clinicopathological variables, and glioma patient outcome. METHODS: Immunohistochemical staining was performed to detect the expression patterns of EphrinB2 and EphB4 in the biopsies from 96 patients with primary gliomas. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of patients. RESULTS: Immunohistochemical analysis revealed that the expression of EphrinB2 was significantly correlated with that of EphB4 (r=0.86, p=0.002). EphrinB2 and EphB4 were significantly associated with the Karnofsky performance scale (KPS) score and World Health Organization grades of patients with gliomas, respectively. Especially, the positive expression rates of EphrinB2 and EphB4 were significantly higher in patients with higher grade (both p=0.001) and lower KPS score (p=0.002 and 0.003, respectively). Multivariate Cox regression analysis revealed that EphrinB2 and EphB4 expressions were both independent prognostic factors for progress-free survival of glioblastoma patients (both p=0.02). CONCLUSION: Our data indicated for the first time that EphrinB2 and EphB4 expressions increase according to the histopathological grade and KPS score of glioma, and their expression levels are related to the progression-free survival of glioblastoma patients.