Volume 42, Issue 3 (9-2018)
Research in Medicine 2018, 42(3): 182-188 |
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Nourbazargan H, Nadji A, Mirab Samiee S, Paryan M, Mohammadi-Yeganeh S. Design and development of TaqMan Real-time PCR assay for detection and viral load determination of HIV-1. Research in Medicine 2018; 42 (3) :182-188
URL: http://pejouhesh.sbmu.ac.ir/article-1-1855-en.html
URL: http://pejouhesh.sbmu.ac.ir/article-1-1855-en.html
, smyeganeh@gmail.com
Abstract: (4923 Views)
Background: Human Immunodeficiency Virus Type 1 (HIV-1) is one of the most important blood-borne
infectious viruses that are considered a global problem, thus it is important to diagnose it with high accuracy
and sensitivity. Serologic methods do not adequately detect this infection. Therefore, the purpose of the
present study was to design a sensitive method based on TaqMan Real-time PCR method for diagnosis of
HIV-1.
Materials and Methods: Primers and probes were designed using bioinformatics softwares for a region of
200 pairs of HIV-1 INT gene. The sequence was cloned into T/A cloning vector and in-vitro RNA transcription
was performed to prepare standards for analytical sensitivity assay. To determine the analytical specificity,
NCBI BLAST and different viral and bacterial samples were used. Clinical specificity was determined using
negative plasma samples.
Results: The method introduced was able to detect as low as 10 copies of HIV-1 RNA/ml. Furthermore, it
was linear in the range of 10-109 copies/ml. By examining the negative samples, the specificity of this method
was determined to be 100%. Intra- and Inter-assay results ranged from 0.3% to 2.5% and 0.7% to 4.5%,
respectively, that showed high reproducibility of the assay.
Conclusion: Due to proper sensitivity and specificity, rapid analysis, being user-friendly, and relatively
low cost, as compared with commercial kits, the method introduced in the present study can be suitable to
accurately diagnose HIV-1 virus. Applying this in-house Real-time PCR assay, viral infection can also be
detected before seroconversion and appearance of bloodstream antibodies, which can reduce window period
of this infection.
infectious viruses that are considered a global problem, thus it is important to diagnose it with high accuracy
and sensitivity. Serologic methods do not adequately detect this infection. Therefore, the purpose of the
present study was to design a sensitive method based on TaqMan Real-time PCR method for diagnosis of
HIV-1.
Materials and Methods: Primers and probes were designed using bioinformatics softwares for a region of
200 pairs of HIV-1 INT gene. The sequence was cloned into T/A cloning vector and in-vitro RNA transcription
was performed to prepare standards for analytical sensitivity assay. To determine the analytical specificity,
NCBI BLAST and different viral and bacterial samples were used. Clinical specificity was determined using
negative plasma samples.
Results: The method introduced was able to detect as low as 10 copies of HIV-1 RNA/ml. Furthermore, it
was linear in the range of 10-109 copies/ml. By examining the negative samples, the specificity of this method
was determined to be 100%. Intra- and Inter-assay results ranged from 0.3% to 2.5% and 0.7% to 4.5%,
respectively, that showed high reproducibility of the assay.
Conclusion: Due to proper sensitivity and specificity, rapid analysis, being user-friendly, and relatively
low cost, as compared with commercial kits, the method introduced in the present study can be suitable to
accurately diagnose HIV-1 virus. Applying this in-house Real-time PCR assay, viral infection can also be
detected before seroconversion and appearance of bloodstream antibodies, which can reduce window period
of this infection.
Type of Study: Original |
Received: 2018/02/16 | Accepted: 2018/07/22 | Published: 2018/10/1
Received: 2018/02/16 | Accepted: 2018/07/22 | Published: 2018/10/1
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