c-DNA of HIV-1 detection on spot of Buffy-Coat of leukocytes (DBCS)
AbstractIntroduction:The elective way for the diagnosis of HIV-1-infection in the window period and in children under the age of 16-18 months is to search virus integrated in leukocytes. Aim of the study was to assess the sensitivity and specificity of extraction from Buffy-Dried Coat Spot (DBCS) in leukocyte to detect c-DNA with nested-PCR in HIV-1-infected individuals compared to Dried Blood Spot (DBS) both extracted by automated instrument EZ1 (QIAGEN, Hilden, Germany). Both DBCS and both DBS were compared with those tests from whole blood by conventional DNA-extraction Methods: Five ml of whole blood from 50 HIV-infected individuals were collected. 40 μl of each sample were spotted on “FTA ELUTE Micro Card” (Whatman, Inc., Clifton, NJ), 200 μl were extracted according to the manual procedure (QIAGEN “QIAamp DNA minikit) and the remaining sample was incubated at 37 °C for 120 minutes. Plasma was centrifuged at 1000 rcf/1g for 10 minutes at room temperature. Forty μl of the obtained buffy-coat was spotted. Both DBCS and both DBS were dried at room temperature for 24 hours.Two of 5 punch from each spot were extracted with TISSUE DNA kit (Biorobot EZ1 DSP “Qiagen”) and eluted in 50 μl of buffer.The recovery of genomic DNA was measured amplifying the ß-globin gene by Real-Time “SybrGreen I”.The DNA was amplified for the “pol” gene of HIV-1 by nested PCR and revealed in “SYBR-green I”. Eight HIV-antibody-negative samples were used as internal control. Results:The experimental protocol adopted for the DBCS showed high sensitivity and specificity.The extracted DNA from DBS and DBCS was characterized by excellent quality and without any inhibitory agents. The amount of proviral DNA extracted from DBCS is similar to that obtained by conventional extraction, while the DBS test was significantly less sensitive. Conclusion:These preliminary data suggest that the amount of c-DNA obtained with DBS technique is often not enough for the diagnosis of HIV-1. Recovery of HIV-1 proviral c-DNA obtained by DBCS assay seems to provide higher sensitivity compared to the DBS-based extraction procedure.Furthermore seems to be equivalent to the whole blood manual extraction yield.Additionally, the SybrGreen I detection system shows a similar sensitivity and specificity compared to the traditional agarose gel detection and appears to be less expensive.
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Copyright (c) 2010 Marco Rossi de Gasperis, Maria Daniela Caione, Carlo Concato, Ersilia Fiscarelli, Nicola Di Pietro, Vittorio Salotti, Lorenza Putignani, Donato Menichella, Francesco Callea
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