A Molecular Approach to Nested RT-PCR Using a New Set of Primers for the Detection of the Human Immunodeficiency Virus Protease Gene.
BACKGROUND
The human immunodeficiency virus (HIV-1) is the etiologic agent of AIDS. The disease can be transmitted via blood in the window period prior to the development of antibodies to the disease. Thus, an appropriate method for the detection of HIV-1 during this window period is very important.


OBJECTIVE
This descriptive study proposes a sensitive, efficient, inexpensive, and easy method to detect HIV-1.


METHODS
In this study 25 serum samples of patients under treatment and also 10 positive and 10 negative control samples were studied. Twenty-five blood samples were obtained from HIV-1-infected individuals who were receiving treatment at the acquired immune deficiency syndrome (AIDS) research center of Imam Khomeini hospital in Tehran. The identification of HIV-1-positive samples was done by using reverse transcription to produce copy deoxyribonucleic acid (cDNA) and then optimizing the nested polymerase chain reaction (PCR) method. Two pairs of primers were then designed specifically for the protease gene fragment of the nested real time-PCR (RT-PCR) samples. Electrophoresis was used to examine the PCR products. The results were analyzed using statistical tests, including Fisher's exact test, and SPSS17 software.


RESULTS
The 325 bp band of the protease gene was observed in all the positive control samples and in none of the negative control samples. The proposed method correctly identified HIV-1 in 23 of the 25 samples.


CONCLUSIONS
These results suggest that, in comparison with viral cultures, antibody detection by enzyme linked immunosorbent assay (ELISAs), and conventional PCR methods, the proposed method has high sensitivity and specificity for the detection of HIV-1.
Shirvan Center of Higher Health Education, North Khorasan University of Medical Sciences, Bojnurd, IR Iran.
Mohammad
M
Zarei
Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
Mehrdad
M
Ravanshad
Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
Ashraf
A
Bagban
Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
Shahab
S
Fallahi
HIV Protease; HIV-1; Nested PCR
J Virol. 2007 Oct;81(19):10209-19
17634242
J Acquir Immune Defic Syndr. 2002 Feb 1;29(2):184-90
11832690
Science. 1988 Nov 25;242(4882):1171-3
2460925
Nature. 1995 Jan 12;373(6510):123-6
7816094
Nat Med. 2009 Aug;15(8):871-2
19648927
J Mol Biol. 2007 Jun 15;369(4):1029-40
17467738
J Clin Microbiol. 2001 Aug;39(8):2937-45
11474017
Nat Biotechnol. 2000 Feb;18(2):199-204
10657128
Methods Mol Biol. 2002;193:65-79
12325526
Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6900-3
7688465