DESCRIPTION OF TECHNIQUES TO BE USED
This part obviously depends greatly on the type of study. You need to think of the following:
Here is a TB project describing methods.
AIM 2: Establish the feasibility of using the Q-PCR assay in PBMCs to distinguish active TB cases from non-infected healthy controls. Preliminary screening to evaluate the sensitivity and specificity of the Q-PCR assays in human PBMCs will be performed in individuals with a confirmed diagnosis of pulmonary TB as positive controls, and in healthy individuals as negative controls. TB patients will be prospectively identified at the TB clinic from HCHD, explained about the study, and invited to participate at the time they are following the routine collection of three morning sputa for direct smear and culture. Blood will be collected at this time, prior to the initiation of chemotherapy. Confirmed diagnosis of pulmonary TB will be based on a MTB-positive sputum by direct smear, culture, and/or the clinical criteria. Based on the average number of patients that HCHD identifies each year, we anticipate that in the project period we will examine at least 10 individuals with confirmed TB. Controls will be healthy PPD-positive students identified at the University of Texas-Brownsville during registration (see letter from Dr. Iracheta). Blood will be collected from the individuals that voluntarily decide to participate and sign the informed consent (appendix).
Blood specimen processing. Buffy coat and plasma will be separated from EDTA-treated blood samples, an aliquot will be saved at -70°C for MTB culture in case the PCR is positive. DNA extraction will be performed according to the protocols developed in the first specific aim. If necessary, the technique will be further refined for optimization.
Q-PCR and MTB culture: Based on the findings for guinea pig, the Q-PCR protocol described in Aim 1 will be adapted to the human model. A positive PCR is defined as the presence of any detectable quantity of target DNA. Relative quantification will be done using ApoB as an indicator of the number of human genomes in each sample. ApoB has been already used routinely by Dr. Shipley and collaborators for this purpose in humans (unpublished data), and has been standardized in our laboratory as well. We anticipate that the number of host genomes may vary extensively between patient specimens due to variations in the total volume of blood collected and number of white blood cells/ml of blood. Such fluctuations may be particularly evident in HIV/AIDS patients, where the number of white blood cells is frequently diminished. All patients with a positive PCR for DNA will be cultured using BACTEC