Detection of Adenoviruses from clinical samples in bone marrow transplant patients by nested PCR [polymerase chain reaction]

Adenoviruses are recognized as common human pathogens that are responsible for a wide variety of infectious syndromes. Bone marrow transplant patients are prone to life threatening opportunistic infections like adenoviruses. The nested polymerase chain reaction has provided an alternative, sensitive diagnostic method for detection of Adenoviruses. In this study we developed PCR from hexon genes as rapid diagnostic method of Advs infections on different clinical samples. Adenovirus infections was defined as the presence of DNA in the blood, urine, stool, or respiratory lavage from bone marrow transplant patients. Two sets of primers [Group specific primers and internal primers] were required to optimize the PCR protocol. This highly sensitive method could detect different types of Advs in two separate sets of PCR. Therefore, DNA amplification in BMT patients would be valuable screening way to evaluate bone marrow transplant recipients. Early detection of Advs by PCR assay is important to asymptomatic infections or preventing aggressive antiviral therapy

T cell expansion from umbilical cord blood without thymic stroma cells after stimulation with SCF, IL-7, and IL-2

We analyzed in vitro expansion and differentiation of T progenitor cells from umbilical cord blood in the absence of thymic epithelium. The expansion setup is performed in the presence of SCF, IL-7 , and IL -2 with autologous serum .Using CBMCs as initial source , we compared the growth kinetics of several cell populations in either whole CBMC or CD34+ -enriched-, as well as in CD3CD4CD8-depleted expansion assays by FACS analysis. After 11 days of culture, cell increase values were about 7 fold for CD3+, 6 fold for CD3+CD4+, 7 fold for CD3+CD8+, 4fold for CD3+CD56, 6fold for CD56+, and 0.2 fold for CD34+. We characterized the developmental state of these cell populations by RT -PCR analysis of the lymphoid differentiation markers RAG-1 and pre T-Alpha. In all samples , transcripts of both markers could be detected from day 0 though day 11, however , in case of pre - T-Alpha, nested PCR was always required , indicating lower expression . These findings; therefore, demonstrate that T-cell differentiation events [as opposed to mere expansion] do occur in stroma cell free expansion assays

development a polymerase chain reaction [PCR] to detect C.pneumoniae and C.psittaci

C.pneumoniae and C.psittaci both cause respiratory infections in human detection of these organisms in tissue culture is difficult and serological testing is unreliable. There are no sensitive and reliable tests for the detection of these organisms. The polymerase chain reaction [PCR ] has provided an alternative diagnostic method for the detection of these fastidious organisms. The aim of this study was to develop a PCR to detect S.pneumonia and C.psittaci from clinical samples. The PCR was optimized in a series of experiments. To determine if the optimized PCR could be applied to clinical samples, mock positive specimen were produced by adding chlamydiae to throat swab from healthy adults. The DNA was extracted by phenol/chloroform. When tested by PCR all the throat swabs were negative. However, when diluted and retested, many of the swabs were positive and 10 IFU of C.psittaci and 100 IFU of S.pneumonia could be detected. This experiment indicates that inhibitor to PCR are found in throat swab and further work is needed on specimen preparation. The PCR was optimized in a series of experiments. The optimal conditions were to use a two segment PCR with 68 degreeC annealing and polymerization temperature, 2.0mM Mgcl2, 0.2microM primers and 40 cycles. The PCR was highly sensitive and could detect one inclusion forming unit with both S.pneumonia and C.psittaci strains. Two human strains and one nonhuman strain of S.pneumonia and two avian strains and three mammalian strains of C.psittaci were used to determine the specificity of PCR. The PCR detected all these different strains. C.trachomatis strains were not detected. Various bacterial strains, fungi DNA, and human DNA were negative in PCR and no amplification DNA was found in negative controls