Teknik reverse transcription – polymerase chain reaction (RT-PCR) dan hibridisasi dot blot dengan pelacak DNA untuk deteksi human immunodeficiency virus (HIV) dalam serum darah

Main Article Content

Maria Lina Rosilawati
Budiman Bela

Abstract

LATAR BELAKANG
Teknik biologi molekuler seperti teknik reverse transcription – polymerase chain reaction (RT-PCR) dot blot hybridization dengan pelacak DNA berlabel biotin dapat mendeteksi human immunodeficiency virus (HIV) dalam serum darah. Teknik ini selanjutnya dapat diterapkan untuk skrining HIV donor jaringan biologi terutama dari Bank Jaringan Riset Badan Tenaga Nuklir Nasional (BATAN), seperti amnion, allograft steril radiasi, melalui darahnya. Penelitian ini bertujuan untuk membandingkan sensitivitas metode RT-PCR hibridisasi dot blot dengan pelacak DNA berlabel biotin dan RT-PCR elektroforesis gel agarosa untuk deteksi HIV.

METODE
Penelitian ini menggunakan serum darah dari Rumah Sakit Ketergantungan Obat (RSKO) Fatmawati. Jumlah serum yang dipakai sebanyak 55 sampel terdiri dari 5 sampel negatif HIV hasil uji serologi dengan rapid test dan 50 sampel dengan enzyme linked immunoassay (ELISA). Ekstraksi RNA HIV sampel darah dilaksanakan menggunakan kit RNA viral extraction sedangkan teknik one step RT-PCR digunakan untuk amplifikasi DNA.

HASIL
Hasil penelitian menunjukkan pada 55 sampel yang diuji baik dengan teknik RT-PCR elektroforesis gel agarosa maupun RT-PCR hibridisasi dot blot, 43 sampel positif mengandung HIV. Hasil RT-PCR hibridisasi dot blot jauh lebih jelas dibanding dengan RT-PCR-elektroforesis gel agarosa. Hal ini terlihat munculnya dot hitam tebal pada film sedangkan pada gel agarosa pita DNA tampak tipis untuk beberapa sampel positif HIV yang sama

KESIMPULAN
Teknik RT-PCR hibridsasi dot blot dengan pelacak DNA berlabel biotin lebih sensitif dibanding dengan RT-PCR elektroforesis gel agarosa untuk mendeteksi HIV.

Article Details

How to Cite
Rosilawati, M. L., & Bela, B. (2007). Teknik reverse transcription – polymerase chain reaction (RT-PCR) dan hibridisasi dot blot dengan pelacak DNA untuk deteksi human immunodeficiency virus (HIV) dalam serum darah. Universa Medicina, 26(3), 111–119. https://doi.org/10.18051/UnivMed.2007.v26.111-119
Section
Review Article

References

O’brien WA, Pomerantz RJ. HIV infection and associated disease. In: Nathanson N, Ahmed R, Scarano FG, Griffin DE, Holmes KV, Murphy FA, et al, editors. Viral pathogenesis. Philadelphia: Lippincott-Raven Publisher; 1997. p. 815-36.

World Health Organization / UNAIDS. AIDS Epidemic Update, Report on the global AIDS epidemic. Last updated June 28, 2007. Available at: http://www.avert.org/aroundworld.htm. Accessed July 1, 2007.

Departemen Kesehatan Republik Indonesia. Peringatan Hari AIDS Sedunia 2006. “Stop AIDS: Saatnya Melayani!!”. Available at: http://www.depkes.go.id/index.php?option= news&task=viewarticle&sid=2353. Accessed November 29, 2006.

Indo IDI. HIV AIDS, 2005. Available at: http://www.idionline.org/infoidi isi.php?news id=946. Accessed April 26, 2006.

World Health Organization / UNAIDS. HIV assay: operational characteristics (Phase1) Report 15 Antigen/Antibody ELISAs; 2004.

Kleinmans S, Busch MP, Hall L, Thomson R, Glynn S, Galahan D, et al. False positive HIV-1 test result in a low risk screening setting of voluntary blood donation. JAMA 1998; 280: 1080-5.

Lane HC. Human immunodeficiency virus. In : Rose NR, Macario EC, Folds JD, Lane HC, Nakamura RM, editors. Manual of Clinical Laboratory Immunology. 5th ed. Washington D.C.: ASM Press; 1997. p. 763-809.

Abravaya K, Esping C, Hoenle R, Gorzowski J, Perry R, Kroeger P, et al. Performance of a multiplex qualitative PCR LCx assay for detection of human immunodeficiency virus type 1 (HIV-1) group M subtypes, group O, and HIV-2. J Clin Microbiol 2000; 38: 716-23.

Goldsby RA, Kindt TJ, Osborne BA. AIDS and other immunodeficiencies. In: Kuby Immunology. 4th ed. New York: W.H Freeman and Company. 2000. p. 485.

Legge A. Direct HIV testing of donated blood is inevitable. BMJ 1997; 314: 1433.

Lackritz EM, Satten GA, Aberle-Grasse J. Estimated risk of transmission of human immunodeficiency virus by screened blood in the United States. N Engl J Med 1995; 333: 1721-5.

Fideler BM, Vangsness Jr CT, Moore T, Li Z, Rasheed S. Effect of gamma irradiation on the human immunodeficiency virus. J Bone Joint Surg 1994; 76-A: 1032-5.

Salzman NP, Psallidopoulos M, Prewett AB. Detection of HIV in bone allografts prepared from AIDS autopsy tissue. Clin Orthop 1993; 292: 384.

Simonds RJ, Holmberg SD, Hurwitz RL, Coleman TR, Bottenfield SRN, Conley LJ, et al. Transmission of human immunodeficiency virus type I from a seronegative organ and tissue donor. N Engl J Med 1992; 326: 726-32.

Dickover RE, Herman SA, Saddiq K, Wafer D, Dillon M, Bryson YJ. Optimization of specimen-handling procedures foraccurate quantitation of level of human immunodeficiency virus RNA in plasma by reverse transcriptase PCR. J Clin Microbiol 1998; 36: 1070-3.

Sebire K, McGavin K, Land S, Middleton T, Birch C. Stability of human immunodeficiency virus RNA in blood specimens as measured by a commercial PCR-based assay. J Clin Microbiol 1998; 36: 493-8.

Kwok, Sninsky JJ. PCR detection of human immunodeficiency virus type I proviral DNA sequences. In: Persing DH, Smith TF, Tenover FC, White TJ, editors. Diagnostic molecular microbiology principles and applications. Washington D.C.: American Society for Microbiology; 1993. p. 309-15.

Holodniy M, Mole L, Yen-Lieberman B, Margolis D, Starkey C, Caroll R, et al. Comparative stabilities of quantitative human immunodeciency virus RNA in plasma from samples collected in Vacutainer CPT, vacutainer PPT, and standard vacutainer tubes. J Clin Microbiol 1995; 35: 1562-6.

Elbeik T, Nassos P, Kipnis P, Haller B, Ng VL. Evaluation of the vacutainer PPT plasma preparation tube for use with the Bayer Versant assay for quantification of human immunodeficiency virus type 1 RNA. J Clin Microbiol 2005; 43: 3769-71.

Lew J, Reichelderfer P, Fowler M, Bremer J, Carrol R, Cassol S, et al. Determination of levels of human immunodeficiency virus type 1 RNA in plasma: reassessment of parameters affecting assay outcome. J Clin Microbiol 1998; 36: 1471-9.

Fatmawati DN. Reverse transcription polymerase chain reaction (RT-PCR) sebagai uji alternatif untuk deteksi infeksi human immunodeficiency virus type 1 (HIV-1) (Tesis). Jakarta: Program Pendidikan Dokter Spesialis I, Fakultas Kedokteran, Universitas Indonesia; 2006.

Anonymous. Human immunodeficiency virus (AIDS). Bremancos Diagnostic. Inc. Available at: http://www.bremancos.com. Accessed June 19, 2004.

Mylonakis E, PaLiou M, Lally M, Flanigan TP, Rich JD. Laboratory testing for infection with the human immunodeficiency virus: established and novel approaches. Am J Med 2000; 109: 568-76.

Pasquier C, Sandres-Saune K, Mansury JM, Puissant B, Viraben R, Spenato N, et al. Virological exploration of individuals with discordant HIV screening tests. J Clin Virol 2004; 30: 218-23.

Gasasira AF, Dorsey G, Kamya MR, Havlir D, Kiggundu M, Rosenthal PJ, et al. False-positive results of enzyme immunoassay for human immunodeficiency virus in patient with uncomplicated malaria. J Clin Microbiol 2006; 44: 3021-4.

Hilmy N, Lina M. Effect of ionizing radiation on viruses, proteins and prions. In: Phillips GO, von Versen R, Strong DM, Nather A, editors. Advances in tissue banking. Singapore: World Scientific Publishing Co. Pte. Ltd. 2001. p. 358-75.

International Atomic Energy Agency (IAEA). Organization of radioisotopes based molecular biology laboratory (IAEA-TECDOC-1528). Vienna, Austria: IAEA: December 2006.