American Journal of Laboratory Medicine

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

Received: Jan. 29, 2017    Accepted: Feb. 21, 2017    Published: Mar. 07, 2017
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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.

DOI 10.11648/j.ajlm.20170201.12
Published in American Journal of Laboratory Medicine ( Volume 2, Issue 1, January 2017 )
Page(s) 7-12
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

HIV-1, DNA Extraction, Multiplex PCR, Real Time PCR

References
[1] WHO. Early detection of HIV infection in infants and children. Guidance note on the selection of technology for the ealy diagnosis of HIV in infants and children. WHO, Geneva, Switzerland, 2007. Available online at http://www.who.int/hiv/paediatric/EarlydiagnostictestingforHIVVer_Final_May07.pdf
[2] Department of Disease Control. Thailand National Guidelines on HIV/AIDS Treatment and Prevention 2014. Department of Disease Control, Ministry of Public Health, Nonthaburi, Thailand. Available online at http://www.silomclinic.in.th/file/2014ThailandHIVguidelines.pdf.
[3] Warachit P, Wongsiree S, Sanguanwong S, Ruchusatwat N, Uneklabh C. Detection for Proviral DNA of HIV-1 by Nested PCR. Bulltin of the Department of Medical Sciences.1994; 36: 11-18.
[4] Jan A, Eva MF. Simple, sensitive and specific detection of Human Immunodeficiency Virus Type 1 in clinical specimens by Polymerase Chain Reaction with Nested Primers. J Clin Microbiol. 1990; 28: 1560-1564.
[5] Wei L, Hua Y, Kimberly R, Chou-Pong Pau and Chin-Yih Ou. Detection of Human Immunodeficiency Virus type 1 DNA in dried blood spots by a Duplex Real-Time PCR assay. J Clin Microbiol. 2005; 43(4): 1851-1857.
[6] Sarah ML, Anne BM, Caroline CC, Anangisye IM, Sally E, Ben A, David JS, Lorenz VS, Werner S, Wendy SS, John AB, John AC. Evaluation of a dried blood spot HIV-1 RNA program for early infant diagnosis and viral load monitoring at rural and remote health care facilities. AIDS. 2009; 23(18): 2459-2466.
[7] Sujit RJ, Douglas HY, Sally Mm, David MK. Rapid, point-of-care extraction of Human Immunodeficiency Virus Type-1 proviral DNA from whole blood for detection by Real-Time PCR. J Clin Microbiol. 2009; 47(8): 2363-2368.
[8] International Laboratory Accreditation Cooperation (ILAC). ISO 15189 Medical Laboratory accreditation. The ILAC Secretariat, NSW 2128, Australia. 2011. Available online at https://www.ukas.com/services/accreditation-services/medical-laboratory-accreditation-iso-15189/
[9] Busarawan S, Nuanchawee W, Supaporn C, Noppavan J, Wiyada C. A study to implement early diagnosis of HIV infection in infants born to infected mothers. Southeast Asian Journal of Tropical Medicine and Public Health. 2003; 34(3): 221-226.
[10] Fischer A, Lejczak C, Lambert C, Servais J, Makombe N, Rusine J, Staub T, Hemmer R, Schneider F, Schmit JC, Arendt V. Simple DNA extraction method for dried blood spots and comparison of two PCR assays for diagnosis of vertical Human Immunodeficiency Virus type 1 transmission in Rwanda. Journal of Clinical Microbiology. 2004; 42(1): 16-20.
[11] Sally MM, Robin LW, Sujit RJ, Douglas HY, Diana H, David MK. A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR. Journal of Viral Methods. 2015; 214: 37-42.
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  • APA Style

    Athicha Mahayotha, Watjana Changthong, Rassame Aomsin, Kantanakon Poiyim. (2017). Evaluating the HIV-1 Proviral DNA Detection by Use of Real Time PCR from Blood Samples and Dried Blood Spots. American Journal of Laboratory Medicine, 2(1), 7-12. https://doi.org/10.11648/j.ajlm.20170201.12

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    ACS Style

    Athicha Mahayotha; Watjana Changthong; Rassame Aomsin; Kantanakon Poiyim. Evaluating the HIV-1 Proviral DNA Detection by Use of Real Time PCR from Blood Samples and Dried Blood Spots. Am. J. Lab. Med. 2017, 2(1), 7-12. doi: 10.11648/j.ajlm.20170201.12

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    AMA Style

    Athicha Mahayotha, Watjana Changthong, Rassame Aomsin, Kantanakon Poiyim. Evaluating the HIV-1 Proviral DNA Detection by Use of Real Time PCR from Blood Samples and Dried Blood Spots. Am J Lab Med. 2017;2(1):7-12. doi: 10.11648/j.ajlm.20170201.12

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  • @article{10.11648/j.ajlm.20170201.12,
      author = {Athicha Mahayotha and Watjana Changthong and Rassame Aomsin and Kantanakon Poiyim},
      title = {Evaluating the HIV-1 Proviral DNA Detection by Use of Real Time PCR from Blood Samples and Dried Blood Spots},
      journal = {American Journal of Laboratory Medicine},
      volume = {2},
      number = {1},
      pages = {7-12},
      doi = {10.11648/j.ajlm.20170201.12},
      url = {https://doi.org/10.11648/j.ajlm.20170201.12},
      eprint = {https://download.sciencepg.com/pdf/10.11648.j.ajlm.20170201.12},
      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.},
     year = {2017}
    }
    

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  • TY  - JOUR
    T1  - Evaluating the HIV-1 Proviral DNA Detection by Use of Real Time PCR from Blood Samples and Dried Blood Spots
    AU  - Athicha Mahayotha
    AU  - Watjana Changthong
    AU  - Rassame Aomsin
    AU  - Kantanakon Poiyim
    Y1  - 2017/03/07
    PY  - 2017
    N1  - https://doi.org/10.11648/j.ajlm.20170201.12
    DO  - 10.11648/j.ajlm.20170201.12
    T2  - American Journal of Laboratory Medicine
    JF  - American Journal of Laboratory Medicine
    JO  - American Journal of Laboratory Medicine
    SP  - 7
    EP  - 12
    PB  - Science Publishing Group
    SN  - 2575-386X
    UR  - https://doi.org/10.11648/j.ajlm.20170201.12
    AB  - 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.
    VL  - 2
    IS  - 1
    ER  - 

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Author Information
  • Advanced Molecular Detection Laboratory (AMD Lab.), Regional Medical Sciences Center 7 (RMSc 7), Department of Medical Sciences, Ministry of Public Health, Khon Kaen, Thailand

  • Advanced Molecular Detection Laboratory (AMD Lab.), Regional Medical Sciences Center 7 (RMSc 7), Department of Medical Sciences, Ministry of Public Health, Khon Kaen, Thailand

  • Advanced Molecular Detection Laboratory (AMD Lab.), Regional Medical Sciences Center 7 (RMSc 7), Department of Medical Sciences, Ministry of Public Health, Khon Kaen, Thailand

  • Advanced Molecular Detection Laboratory (AMD Lab.), Regional Medical Sciences Center 7 (RMSc 7), Department of Medical Sciences, Ministry of Public Health, Khon Kaen, Thailand

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