Evaluating the HIV-1 Proviral DNA Detection by Use of Real Time PCR from Blood Samples and Dried Blood Spots

Abstract

The aim of this study is to evaluate the HIV-1 Infection Examination by using Real time PCR method from blood samples and Dried Blood Spots (DBS) in order to evaluate the efficiency and readiness of the method. The experimental complies with the requirements of the quality testing standard ISO 15189 which includes the following: comparing the infection examination results of HIV-1 between Real time PCR method and Multiplex Nested PCR method from the blood samples collected from children during clinical routines. Comparing the infection examination results of HIV-1 using Real time PCR method from dried blood spots which already had test results from the Multiplex Nested PCR method and improve DNA extraction by dissolving blood with 5% Chelex-100 as well as DNA extraction by dissolving blood with MagNa Pure Compact kit. Comparing the test results with DNA extraction using QIAamp and test to find the lowest amount of HIV-1 DNA in DBS. This extraction is done by using an improved method which is Real time PCR. By using the Real Time PCR method provided positive Ct criteria of ≤ 37, it was discovered that 22 samples tested positive and 436 tested negative, and is in compliance with the Multiplex Nested PCR Method. DNA from 140 Dried Blood Spot Samples, which were extracted using an improved method, provided a positive Ct criteria of ≤ 37. It was discovered that only 26 out of 40 samples (75%) tested positive. As for the negative group sample, it was discovered that 100 samples (100%) were tested negative. When the deciding Ct criteria was adjusted to ≤ 40, it was discovered that all 40 samples (100%) were positive and 100 samples (100%) were negative. When comparing the examination results of these samples, which were extracted with QIAamp DNA Mini Kit, were 100% match. In addition the limit of detection of the improved method for HIV-1 DNA is ≥ 250 copies/ml. These results show that the Infection Examination HIV-1 from blood by using Real time PCR provides results that were not different from the Multiplex Nested PCR. As for the Exanimation using DBS, the criteria for deciding positive results using Real time PCR does have an effect on the speed and accuracy of the examination. Due to the fact that the samples began with different amounts, using different criteria for positive results should be considered.