Preliminary evaluation of miR-1307-3p in human serum for detection of 13 types of solid cancer using microRNA chip.

Early detection and treatment are crucial for increasing the five-year survival rates of various cancers. Low-cost and convenient cancer screening tests are also critically important. Circulating microRNAs are reported as potential biomarkers for various cancers. Recently, miR-1307-3p was found to be a cancer-related microRNA. We evaluated the expression levels of miR-1307-3p in sera obtained from 254 patients with thirteen types of cancer (colon cancer, lung cancer, gastric cancer, liver cancer, bladder cancer esophageal cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, biliary tract cancer, brain cancer, sarcoma) and 27 non-cancer samples using isothermal amplification and microRNA chip. The expression levels of miR-1307-3p in sera obtained from cancer patients were clearly different from those obtained from non-cancer samples and differentiated the validation cohort into cancer patients and non-cancer control with high accuracy (AUC: 0.98; sensitivity: 0.98; specificity: 0.85). These results showed the potential relevance of miR-1307-3p in serum for the development of new diagnostic examination tools for cancer patients.

Multiplex Real-Time Loop-Mediated Isothermal Amplification Using an Electrochemical DNA Chip Consisting of a Single Liquid-Flow Channel.

We developed a multiplex system capable of simultaneously quantifying different target sequences by applying an electrochemical DNA chip that consists of single liquid-flow channel with primers designed for loop-mediated isothermal amplification (LAMP). We applied this system for detecting mature microRNAs (miRNAs). miRNAs extracted from serum were enzymatically lengthened to about 100 base pairs by reverse-transcription and elongation reactions. The LAMP primers for amplifying the lengthened miRNAs were adsorbed and immobilized on the surface of the liquid-flow channel at five different positions. A LAMP solution containing the lengthened miRNAs, Tin DNA polymerase, and ruthenium hexaamine (RuHex) as a redox compound was injected into the DNA chip. The electrochemical reaction of RuHex in the LAMP solution was then measured continuously via linear-sweep voltammetry at 65 °C. The LAMP reaction of the positive control revealed that the cathodic peak current of RuHex increased. Additionally, the initial number of miRNA copies was correlated with the time when the cathodic current began to increase. Five miRNAs were simultaneously detected at 103-106 copies per 50 µL within 2 h. We expect these results will be useful for developing a simple and stable electrochemical-based method for the real-time monitoring of miRNAs, while also facilitating the implementation of electrochemical DNA chips for molecular analyses.

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip.

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.

A novel voltammetric approach for real-time electrochemical detection of targeted nucleic acid sequences using LAMP.

We have developed a novel voltammetric DNA chip for real-time electrochemical detection of targeted nucleic acid sequences using loop-mediated isothermal amplification (LAMP) and ruthenium hexaamine (RuHex) as the intercalative redox compound. A GspSSD DNA polymerase was used for LAMP owing to its tolerance of the intercalative redox compound. The electrochemical reaction of 1 mM RuHex in the LAMP solution was measured continuously by linear sweep voltammetry at 65 °C using an electrochemical DNA chip. According to the LAMP reaction of the positive sample, the cathodic peak current of RuHex increased and the cathodic peak potential of RuHex shifted to negative voltage. The initial number of copies of the targeted nucleic acid was correlated with both the time when the cathodic current began to increase and the time when the cathodic potential began to shift rapidly. 103 to 106 copies/50 µL of the targeted nucleic acid were detected quantitatively and the detection limit was 101 copies within an hour. We expect these results to lead to the realization of simple and stable electrochemical real-time monitoring of targeted nucleic acids while also facilitating the implementation of electrochemical DNA chips in molecular testing.

Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens.

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.

Identification and functional analysis of a new phosphorylation site (Y398) in the SH3 domain of Abi-1.

Abi-1 is an adaptor protein for Abelson kinase (c-Abl), and Abi-1 promotes the Abl-mediated phosphorylation of Mammalian Enabled (Mena) by binding both c-Abl and Mena. Here, we identified a new phosphorylation site (Y398) in the SH3 domain of Abi-1, and disruption of Y398, combined with the previously identified phosphorylation site Y213, significantly weakens the binding of Abi-1 to c-Abl. The SH3 domain of Abi-1 and the proline-rich domain of c-Abl are involved in this interaction. Abi-1 phosphorylation at both sites stimulates the phosphorylation of Mena through the activation of c-Abl kinase. The phosphorylation of Abi-1 also plays a role in enhancing the adhesion of Bcr-Abl-transformed leukemic cells.