hiv-1 is an etiologic agent and a member of the retroviridae family and lentivirus genus ( 1 ) . it also shows rapid turnover and exerts selective immune pressure of the host ( 2 - 4 ) . the p group , which was isolated from a female cameroonian patient , is closely related to immunodeficiency viruses found in gorillas ( 5 ) . group m is responsible for most hiv-1 infections globally and is divided into 10 subtypes ( a - k ) . the most common genetic forms of hiv-1 are subtypes b , a , and c. subtype c is responsible for almost 50% of hiv-1 infections . within group m , the mean genetic diversity of the gag gene is 15% and that of the envelope ( env ) gene is 25% . there are over 20 types of circulating recombinant forms ( crfs ) ( 6 ) , the most common of which are crf01-ae and crf02-ag ( 7 ) . the increasing spread of hiv-1 highlights the urgent need for improved diagnostic methods to detect the infection at an early stage and stem the spread of this disease . currently , the major methods used to detect hiv-1 are enzyme linked immunosorbent assay ( elisas ) , followed by the western blot method ( 8 , 9 ) . during the window period , diagnostic tests based on specific antibodies can not detect the presence of the virus , and serological tests can not diagnose hiv-1 in infants born to infected mothers because of the active transfer of maternal antibodies to the infant ( 10 ) . the development and application of rapid and accurate diagnostic tests and advanced molecular techniques are of the utmost importance . such tests could slow the spread of hiv-1 by speedy detection of infected individuals and products . once detected , the viral load of infected individuals can be measured to determine their prognosis and draw up a treatment plan , in accordance with international standards . the present study describes a sensitive , efficient , and easy method to detect hiv-1 . the study utilized new , highly specific primers for the protease gene site , in combination with nested rt - pcr , to detect hiv-1 subtypes that show extreme nucleotide variation . as the pol gene is a protected region of the virus genome , the primers were designed , as follows : first , the alignment of approximately 1,000 pol sequences was carried out using the gen bank database , and mega4 software ( www.megasoftware.net ) was used to identify protected regions . gene runner software ( http://www.generunner.com ) , an oligo analyzer ( http://eu.idtdna.com ) , and oligo6 ( www.oligo.net ) were used to design the primers . all the primers used in the present study were new and original products developed for use in the present study . the samples were collected from a population of patients infected with hiv-1 . in this study 25 serum samples of patients under treatment and also 10 positive and 10 negative control samples were studied . the study was approved by the bio - safety and medical ethics committee of tarbiat modares university . all samples had been previously confirmed to be positive using the western blot technique , and all were from hiv-1 patients who were receiving treatment . viral rna was extracted using the rna extraction kit ( qiagen , hilden , germany ) . the product of the first round of pcr was 586 bp , and the product of the second round was 325 bp . a 100 bp marker ( dna ladder , fermentas , canada ) was used . to verify the accuracy of the final product , the 25 hiv-1 positive samples the results were then confirmed using the western blot method . to optimize the pcr reaction to detect low levels of the virus in the samples and to obtain the best possible band , using the most appropriate materials and reaction temperatures to create a strong and pure band . the data and results of the positive , negative , and control samples were analyzed using pearson s correlation , regression , and anova analyses and spss17 software . the efficiency of the proposed method was tested on 25 plasma samples from patients infected with hiv-1 , 10 positive serum samples , and 10 negative serum samples . the 325 bp band of the protease gene was clearly observed in all the positive samples after electrophoresis . no band was detected in the negative samples , and there were no nonspecific bands or smears ( figure 1 ) . wells 1 and 2 show the positive samples ; wells 3 and 4 show the negative control samples ; well 5 is the weighted marker . the results of the sequencing of the positive cases further confirmed the accuracy of the proposed method ( table 1 ) . given that the viral load in the hiv-1 patients who were receiving treatment was low , since the discovery of hiv-1 in1984 as the etiologic agent of aids , much progress has been made in identifying this virus . serological tests , including antibody detection , have increased the sensitivity and specificity of the tests . hiv-1 antigen detection tests and cultivation of this virus are useful in some clinical procedures . most cultures , with the exception of those from patients receiving highly active antiretroviral therapy ( haart ) therapy , will be positive within 21 days . the sensitivity of this technique ( cultivation of hiv ) in patients whose blood serum test results are positive is 97% , and the specificity is 100% . the cultivation of hiv is difficult , time - consuming , and less sensitive than pcr . the elisa method , which is based on the detection of the p24 antigen , is also not useful for the detection of hiv-1 because it has low sensitivity . qualitative and quantitative methods can be used for the examination of nucleic acids of a virus . in qualitative methods , the detection of the nucleic acid of the virus is done by rt - pcr . the pcr method can be used to detect the provirus form in peripheral blood monomorphonuclear cells ( pbmc ) cells . this method is useful for the diagnosis of infants born to hiv-1-infected mothers and is more sensitive than virus cultivation and the p24 antigen ( 9 ) . in the present study , rt - pcr is a sensitive technique , and its sensitivity increased using the nested rt - pcr approach . in this study , part of the rt - pcr product was used as a template for the second - round pcr . to avoid amplification of primer dimers and nonspecific products generated in first - round pcr , the nested primers were internal , as compared to the primers used in the first round of the pcr and generated shorter products . as the sequencing of the second - round pcr primers was different from that of the first - round pcr primers , the primer dimers and nonspecific products produced in the first round were not duplicated in the second round . the sensitivity and specificity of the proposed method was greater than those of existing methods . the proposed method can be used to detect viral rna in serum samples with viral loads of less than 100 copies per ml ( 11 ) . hiv-1 transmission through blood and blood products during the window period is of great concern . the application of a sensitive , accurate , and rapid method for the detection of hiv-1 in infants born to infected mothers is essential and requires a method that can identify this stage of infection . the results indicate that the proposed nested rt - pcr method had high sensitivity and specificity , as compared with previous methods . the proposed method is appropriate for use in diagnostic centers as a supplementary technique . in particular , it may be useful for the detection of infection in infants born to hiv-1 infected and the diagnosis of infections in the window period . the results indicate that the proposed nested rt - pcr method had high sensitivity and specificity , as compared with previous methods . the proposed method is appropriate for use in diagnostic centers as a supplementary technique . in particular , it may be useful for the detection of infection in infants born to hiv-1 infected and the diagnosis of infections in the window period .
backgroundthe 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.objectivesthis descriptive study proposes a sensitive , efficient , inexpensive , and easy method to detect hiv-1.patients and methodsin 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.resultsthe 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.conclusionsthese 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 .