[Optimization of loop-mediated isothermal amplification [LAMP] method for detection of herpes simplex virus type 1 and 2]

Type 1 and type 2 herpes simplex [HSV] virus cause infection of central nervous system [encephalitis] in human. The molecular techniques are the best methods for detection of HSV. In this study we evaluated the novel molecular technique of LAMP for detection of HSV-1 and HSV-2. In this experimental study 184 cerebrospinal fluid [CSF] samples were collected from Mofid Hospital. DNA of every sample was extracted by use of Sinagen DNP kit. Based on the HSV DNA polymerase gene, a set of 6 primers were designed and sensitivity and specificity of this method were determined. By adding SYBER Green, LAMP product was identified. The results of LAMP method were compared to those of PCR by chi-square test. Sensitivity of LAMP method determined to be 5 copies/ tube and sensitivity of PCR method determined to be 50 copies/ tube. Both LAMP and PCR methods showed 100% specificity for detection of HSV type 1 and type 2. Among 184 samples, 60 samples were positive by LAMP but 45 samples were positive by PCR method. Sensitivity of LAMP was 10 times higher than that of PCR. Comparison of the results of the two methods by means of chi-square test showed a significant difference [p<0.05]. LAMP method had high sensitivity and specificity for detection of type 1 and type 2 HSV in CSF samples

[Distribution of capsular serotypes in group B streptococci clinical]

Group B Streptococci [GBS] or Streptococcus agalactiae is one of the most common causes of sepsis and meningitis in neonates and of invasive diseases in pregnant women. It can also cause infectious disease among adults with underlying medical conditions like immunocompromised individuals. Polysaccharide capsule is an important virulence factor. Nine GBS serotypes [Ia, Ib, II to VIII] based on capsular polysaccharide antigens have been described. Distribution of capsular serotypes varies over time and by geographic location. The aim of this study was to detect the capsular serotype distribution in GBS clinical isolates based on genotyping of cps-gene cluster and to determine the predominant serotypes of GBS. In this cross sectional study a total of 50 GBS strains were isolated from various clinical sources including: urine, vagina, semen and urethral secretions. GBS was identified by Gram stain, catalase test, CAMP test and also resistance to 0.04 U Bacitracin and SXT disks. DNA was extracted from all the isolates using the wizard SV Genomic DNA Purification system, Promega, USA. The capsular serotype of the isolates was assigned by using a specific-two Multiplex PCR assay. For statistical analysis, Chi-square method was used. SPSS V.13 was also used. In the 50 GBS isolates, the predominant serotypes were III with 25 isolates [50%] and serotype V with 8 isolates [16%]. Seven isolates [14%] belonged to serotype Ia and 7 isolates [14%] belonged to serotype II, respectively. Serotypes Ib, IV, VI, VII and VIII were not found and 3 strains were classified as nontypeable. Based on the results of this study, serotypes III and V were the predominant serotypes in GBS clinical isolates

Evaluation of stimulatory effects of HTLV-I tax protein on CREB and NFKB related signaling pathways two B-glycosidase based reporter plasmids

Human T cell Lymphotropic Virus type I is responsible for Adult T cell Leukemia/Lymphoma and HTLV-I associated myelopathy. The aim of this study was constructing two reporter plasmids to enable us to evaluate the effects of HTLV-I Tax protein upon intra cellular signalling pathways which recruit CREB and NFkB proteins. A complete coding region of bacterial betagalactosidase gene was subcloned into pUC18, followed by inserting a poly adenylation signal downstream to it. Promoter regions of HTLV-I long terminal repeat and Interlukin 2 receptor alpha [which were stimulated by CREB and NFkB respectively] were amplified by PCR and separately inserted upstream to betagalactosidase gene, leading to construction of two reporter plasmids. The effect of cotransfection of a Tax expressing plasmid with each of these plasmids was evaluated by X-gal staining, beta galactosidase ELISA or beta galactosidase activity assay with CPRG substrate. Results clearly showed that both reporter plasmids responded well to stimulation of their promoters by Tax and the produced beta galactosidase could successfully be detected by all three methods. Results of ELISA and assay tests for betagalactosidase showed a high correlation [r=0.949]. Both reporter plasmids constructed in this study are able to produce considerably more betagalactosidase after stimulation by HTLV-I Tax. Advantages and disadvantages of three evaluating method for detection of betagalactosidase are studied and discussed