Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

<p>Abstract</p> <p>Background</p> <p><it>Vibrio parahaemolyticus </it>is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of <it>V. parahaemolyticus</it>. Most <it>V. parahaemolyticus </it>isolates from the environment do not produce TDH or TRH. Total <it>V. parahaemolyticus </it>has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total <it>V. parahaemolyticus </it>using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of <it>Vibrio parahaemolyticus</it>.</p> <p>Results</p> <p>The assay provided markedly more sensitive and rapid detection of <it>V. parahaemolyticus </it>strains than conventional biochemical and PCR assays. The assay correctly identified 143 <it>V. parahaemolyticus </it>strains, but did not detect 33 non-<it>parahaemolyticus Vibrio </it>and 56 non-<it>Vibrio </it>strains. Sensitivity of the LAMP assay for direct detection of <it>V. parahaemolyticus </it>in pure cultures and in spiked shrimp samples was 5.3 × 10²CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 <it>V. parahaemolyticus </it>strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of <it>V. parahaemolyticus </it>required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.</p> <p>Conclusion</p> <p>The LAMP assay is a sensitive, rapid and simple tool for the detection of <it>V. parahaemolyticus </it>and will facilitate the surveillance for control of contamination of <it>V. parahaemolyticus </it>in seafood.</p

Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification

<p>Abstract</p> <p>Background</p> <p><it>Vibrio cholerae </it>is widely acknowledged as one of the most important waterborne pathogen causing gastrointestinal disorders. Cholera toxin (CT) is a major virulence determinant of <it>V. cholerae</it>. Detection of CT-producing <it>V. cholerae </it>using conventional culture-, biochemical- and immunological-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of cholera toxin (CT)-producing <it>Vibrio cholerae</it>.</p> <p>Results</p> <p>The assay provided markedly more sensitive and rapid detection of CT-producing <it>V. cholerae </it>strains than conventional biochemical and PCR assays. The assay correctly identified 34 CT-producing <it>V. cholerae </it>strains, but did not detect 13 CT non-producing <it>V. cholerae </it>and 53 non-<it>V. cholerae </it>strains. Sensitivity of the LAMP assay for direct detection of CT-producing <it>V. cholerae </it>in spiked human feces was 7.8 × 10²CFU per g (1.4 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay for detection of CT-producing <it>V. cholerae </it>required less than 35 min with a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 70 min with human feces from the beginning of DNA extraction to final determination.</p> <p>Conclusion</p> <p>The LAMP assay is a sensitive, rapid and simple tool for the detection of CT-producing <it>V. cholerae </it>and will be useful in facilitating the early diagnosis of human <it>V. cholerae </it>infection.</p

Effect of Sensory Manipulations on Human Joint Stiffness Strategy and Its Adaptation for Human Dynamic Stability

Sensory input plays an important role to human posture control system to initiate strategy in order to counterpart any unbalance condition and thus, prevent fall. In previous study, joint stiffness was observed able to describe certain issues regarding to movement performance. But, correlation between balance ability and joint stiffness is still remains unknown. In this study, joint stiffening strategy at ankle and hip were observed under different sensory manipulations and its correlation with conventional clinical test (Functional Reach Test) for balance ability was investigated. In order to create unstable condition, two different surface perturbations (tilt up-tilt (TT) down and forward-backward (FB)) at four different frequencies (0.2, 0.4, 0.6 and 0.8 Hz) were introduced. Furthermore, four different sensory manipulation conditions (include vision and vestibular system) were applied to the subject and they were asked to maintain their position as possible. The results suggested that joint stiffness were high during difficult balance situation. Less balance people generated high average joint stiffness compared to balance people. Besides, adaptation of posture control system under repetitive external perturbation also suggested less during sensory limited condition. Overall, analysis of joint stiffening response possible to predict unbalance situation faced by human

Field Equations of Massless Fields in the New Interpretation of the Matrix Model

Recently, some of the authors have introduced a new interpretation of matrix models in which covariant derivatives on any curved space can be expressed by large-N matrices. It has been shown that the Einstein equation follows from the equation of motion of IIB matrix model in this interpretation. In this paper, we generalize this argument to covariant derivatives with torsion. We find that some components of the torsion field can be identified with the dilaton and the $B$-field in string theory. However, the other components do not seem to have string theory counterparts. We also consider the matrix model with a mass term or a cubic term, in which the equation of motion of string theory is exactly satisfied.Comment: 21 page

Loops versus Matrices - The nonperturbative aspects of noncritical string

The nonperturbative aspects of string theory are explored for non-critical string in two distinct formulations: loop equations and matrix models. The effects corresponding to D-brane in these formulations are especially investigated in detail. It is shown that matrix models can universally yield a definite value of the chemical potential for an instanton while loop equations can not. This implies that string theory may not be nonperturbatively formulated solely in terms of closed strings.Comment: 58 pages, 3 figures, PTPTeX, published versio

Demonstration of intrahepatic accumulated microbubble on ultrasound represents the grade of hepatic fibrosis

OBJECTIVES: To examine the feasibility of perflubutane-based ultrasound for grading hepatic fibrosis. METHODS: This prospective study included 202 subjects; main study (controls:33, F0–1:35, F2:26, F3:23, cirrhosis:29) and subsequent study (controls:16, F0–1:7, F2:20, F3:7, cirrhosis:6). Diagnostic abilities for assessing fibrosis grade were compared between contrast findings and FIB4 (age × AST/[platelet count × ALT(0.5)]). RESULTS: High-power emission produced an intrahepatic band-like structure, and the three-layer appearance was less frequent and monolayer appearance was more frequent in cirrhosis than controls/chronic hepatitis (P < 0.0001). Intensity difference at 15-min phase showed most significant correlation with fibrosis grade (ρ = 0.79, P < 0.0001), and the best areas under the receiver operating characteristic curves are 0.88 for marked fibrosis, 0.95 for advanced fibrosis and 0.97 for cirrhosis, which were significantly higher than those of FIB4, 0.85 for marked fibrosis, 0.89 for advanced fibrosis and 0.90 for cirrhosis. Sensitivity, specificity and efficiency of the intensity difference were 88%, 72% and 81% for marked fibrosis, 85%, 91% and 89% for advanced fibrosis and 97%, 90% and 91% for cirrhosis, respectively. The subsequent study validated the main study results; significant correlation between the intensity difference and the fibrosis grade (ρ = 0.73–0.77, P < 0.0001). CONCLUSIONS: Perflubutane-based ultrasound accurately predicts the grade of hepatic fibrosis. KEY POINTS: • The behaviour of intrahepatic microbubbles depends on the severity of hepatic fibrosis. • Layer enhancement pattern simply represents the degree of chronic liver disease. • Parenchymal intensity change due to high-power emission predicts the hepatic fibrosis grade

Transplantation of human neural stem/progenitor cells overexpressing galectin-1 improves functional recovery from focal brain ischemia in the mongolian gerbil

Transplantation of human neural stem/progenitor cells (hNSPCs) is a promising method to regenerate tissue from damage and recover function in various neurological diseases including brain ischemia. Galectin-1(Gal1) is a lectin that is expressed in damaged brain areas after ischemia. Here, we characterized the detailed Gal1 expression pattern in an animal model of brain ischemia. After brain ischemia, Gal1 was expressed in reactive astrocytes within and around the infarcted region, and its expression diminished over time. Previously, we showed that infusion of human Gal1 protein (hGal1) resulted in functional recovery after brain ischemia but failed to reduce the volume of the ischemic region. This prompted us to examine whether the combination of hNSPCs-transplantation and stable delivery of hGal1 around the ischemic region could reduce the ischemic volume and promote better functional recovery after brain ischemia. In this study, we transplanted hNSPCs that stably overexpressed hGal1 (hGal1-hNSPCs) in a model of unilateral focal brain ischemia using Mongolian gerbils. Indeed, we found that transplantation of hGal1-hNSPCs both reduced the ischemic volume and improved deficits in motor function after brain ischemia to a greater extent than the transplantation of hNSPCs alone. This study provides evidence for a potential application of hGal1 with hNSPCs-transplantation in the treatment of brain ischemia