ABSTRACT
AIMS: To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus. METHODS AND RESULTS: Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates (n = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10(7)-10(1) CFU ml(-1) and that for tdh2 was 10(7)-10(4) CFU ml(-1). CONCLUSIONS: The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed allows accurate detection of pathogenic V. parahaemolyticus, which is valuable for rapid tracing of infection source during outbreaks.
Subject(s)
Food Microbiology , Nucleic Acid Hybridization/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Seafood/microbiology , Vibrio Infections/diagnosis , Vibrio parahaemolyticus/isolation & purification , Base Sequence , Cloning, Molecular/methods , DNA Primers/genetics , Gene Amplification , Genes, Bacterial , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Sensitivity and Specificity , Vibrio parahaemolyticus/geneticsABSTRACT
BACKGROUND: The long term physiological and radiological outcomes of SARS survivors and their possible determinants are uncertain. METHODS: SARS survivors in a follow up clinic in a regional hospital underwent high resolution computed tomography (HRCT) of the thorax and lung function tests 6 months after admission to hospital. The associations between the clinical and demographic data of the patients and the physiological and radiological outcomes were examined. RESULTS: Fifty seven patients took part in the study. Lung function abnormalities were detected in 43 patients (75.4%), with restrictive defects (n = 16) being most common (28.1%). Radiological abnormalities of any degree were detected in 43 patients (75.4%). Only the use of pulse corticosteroids was associated with the presence of CT abnormalities (p = 0.043, OR 6.65, 95% CI 1.06 to 41.73). CONCLUSIONS: Physiological and radiological abnormalities are still present in a considerable proportion of SARS survivors at 6 months.
Subject(s)
Severe Acute Respiratory Syndrome/diagnostic imaging , Adult , Female , Forced Expiratory Volume/physiology , Humans , Male , Prognosis , Severe Acute Respiratory Syndrome/physiopathology , Tomography, X-Ray Computed/methods , Total Lung Capacity/physiologySubject(s)
Severe Acute Respiratory Syndrome/diagnosis , Severe acute respiratory syndrome-related coronavirus , Humans , Lung/diagnostic imaging , Lung/ultrastructure , Lymphopenia/complications , Microscopy, Electron , Severe acute respiratory syndrome-related coronavirus/ultrastructure , Severe Acute Respiratory Syndrome/diagnostic imaging , Severe Acute Respiratory Syndrome/pathology , Tomography, X-Ray ComputedABSTRACT
Gene amplification and enhanced expression of the epidermal growth factor receptor (EGFR) represent the major molecular genetic alteration in glioblastomas and it may play an essential role in cell growth and in the carcinogenic process. On the other hand, the nuclear suppressor proteins PML and p53 are also known to play critical roles in cancer development and in suppressing cell growth. Here we report that, in glioblastoma cells with defective EGFR function, the expressions of both promyelocytic leukaemia (PML) and p53 were altered. Cells that were transfected with the antisense-cDNA of EGFR were found to have more cells in G1 and fewer cells in S phase. In addition, the transfected cells were found to be non-responsive to EGF-induced cell growth. Interestingly, the expression of the suppressors p53 and PML were found to be significantly increased by immunohistochemical assay in the antisense-EGFR cells. Moreover, the PML expression in many of the cells was converted from the nuclear dot pattern into fine-granulated staining pattern. In contrast, the expressions of other cell cycle regulated genes and proto-oncogene, including the cyclin-dependent kinase 4 (cdk4), retinoblastoma, p16INK4a and p21H-ras, were not altered. These data indicate that there are specific inductions of PML and p53 proteins which may account for the increase in G1 and growth arrest in antisense-EGFR treated cells. It also indicates that the EGF, p53 and PML transduction pathways were linked and they may constitute an integral part of an altered growth regulatory programme. The interactions and cross-talks of these critical molecules may be very important in regulating cell growth, differentiation and cellular response to treatment in glioblastomas.
