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SYBR Green I-based real-time qPCR assays to determine copy number of the IFI16 gene for the detection of Chlamydia trachomatis infection. The IFI16 gene (amplification product amplified by the primers IFI16-F1 and IFI16-R1, respectively) from C. trachomatis in clinical samples of upper and lower ocular samples was investigated. The real-time PCR conditions were 95°C for 5 min and then performed using the following conditions: 95°C for 10 s followed by 40 cycles of 95°C for 5 s and 60°C for 20 s (CFX optical reader, BioRad).
Syber green quantitative real-time PCR (qRT-PCR) to detect the simian immunodeficiency virus (SIV) 1- and 2-specific single copy RNA sequences. This assay is based on the detection of human RNA sequences including HIV-1 RNA species by RT-PCR. During the assay, it is possible to detect the presence an amount of SIV RNA by specific detection of the HIV sequences. The primers used in this assay are: SIV gag primers (SIV1F508 and SIV1R114: template: SIV3B) designed to amplify a 514 bp of the gag region; SIV2F76 (SIV2-F) and SIV2R346 (SIV2-R) designed to detect SIV2-specific sequences less than 533 bp. The virus quantification was performed with TaqMan assay SIV3B and SIV3B-R (primer/probe). Two, three or four replicates of 1/2 dilution series of cDNA from a polytropic strain of SIV-infected primate were analyzed with the SIV quantification assay. qPCR data were analyzed with the iCycler software instrument using the standard curve method. The level of target RNA sequences was extrapolated from standard curve data and expressed as the RNA copy number per 10 pg of starting RNA sample calculated.]
The Ho-gam gene encodes the transmembrane glycoprotein defined by reactive sites for TcI of T. cruzi. The GRAp32 gene lies upstream of this gene and encodes a protein that binds to 400 kDa hexameric glycoconjugates of the parasite, which are described as a major determinant in the invasion apparatus of the parasite. These genes are expressed exclusively in the early stages of T. cruzi infection and may be related to tissue tropism during the sylvatic cycle and establishment of the infection in the mammalian host. These genes may be important markers for both the vector-borne and zoonotic forms of T. cruzi.
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