Analisis Genom HPV 16 Galur UI66 dari Indonesia dan Pengklonaan Fragmen E6/E7 sebagai Kontrol Positif Deteksi HPV 16 = Genome analysis of HPV 16 UI 66 isolate from Indonesia and cloning of E6/E7 fragment as positive control for HPV 16  detection

Dwi Wulandari, author

Abstrak

[HPV 16 merupakan genotipe yang paling sering terdeteksi pada kanker serviks. Berbagai varian HPV 16 telah dikategorikan berdasarkan distribusi geografisnya. Broer et al. telah melaporkan varian HPV 16 di Indonesia berdasarkan analisis gen E6, E7, dan L1. Namun, belum ada laporan mengenai analisis gen-gen lain dari isolat HPV 16 di Indonesia. Metode deteksi dan genotiping merupakan alat utama dalam deteksi infeksi dan evaluasi keberhasilan vaksin HPV. Saat ini, metode deteksi dan genotiping HPV berbasis gen E6/E7 sedang dikembangkan karena gen E6 dan E7 merupakan gen yang paling lestari pada pasien kanker serviks. Standar dan kontrol untuk akurasi deteksi dan genotiping sangat diperlukan. WHO menyediakan standar internasional berupa plasmid rekombinan yang mengandung genom lengkap HPV 16 dan 18 dari materi genetik HPV luar negeri. Oleh karena itu, dalam penelitian ini dilakukan pengklonaan fragmen E6/E7 dari isolat HPV 16 Indonesia yang dapat digunakan sebagai kontrol positif deteksi HPV 16. Fragmen-fragmen dari genom HPV 16 galur UI66 diamplifikasi dengan PCR, disekuensing, kemudian dianalisis variasi genetik pada gen E6, E7, E1, E2, E4, E5, L2, parsial L1, dan LCR, serta dibandingkan dengan referensi HPV 16. Pengklonaan fragmen 1 yang mengandung gen E6/E7 dilakukan dan diuji sebagai kontrol positif deteksi HPV 16 menggunakan PCR dan real-time PCR. Hasil penelitian menunjukkan bahwa tingkat kesamaan nukleotida dan asam amino dari masing-masing gen galur UI66 bervariasi, dengan kesamaan tertinggi berturut-turut adalah pada gen L1 parsial dan protein E7. Kekerabatan masing-masing gen dari galur UI66 sangat bervariasi. Telah diperoleh satu klon yang membawa plasmid rekombinan pUI66F1-E6/E7 yang dapat digunakan sebagai kontrol positif deteksi HPV 16 dengan target gen E6 dan E7.

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HPV 16 is the most commonly detected genotype in cervical cancer. HPV 16 variants have been categorized based on their geographical distribution. Broer et al. have reported HPV 16 variants in Indonesia based on the analysis of E6, E7, and L1 genes. However, there have been no reports on the analysis of other genes from HPV 16 Indonesian isolates. Detection and genotyping methods are primary tools for measuring HPV infection and assessing vaccine efficacy. Currently, detection and genotyping of HPV based on E6/E7 genes are being developed since E6 and E7 are the most conserved genes in cervical cancer patients. Standards and controls for detection and genotyping accuracy are necessary. The WHO provides international standards in the form of a recombinant plasmid containing the complete genome of HPV 16 and 18 from foreign HPV genetic material. Therefore, this research involved cloning fragments of HPV 16 E6/E7 from an Indonesian isolate to be used as a positive control for HPV 16 detection. The genome fragments of HPV 16 UI66 isolates were amplified by PCR, sequenced, and then analyzed for genetic variations in E6, E7, E1, E2, E4, E5, L2, partial L1, and LCR genes, and compared with HPV 16 reference. Cloning of fragment 1, containing E6/E7 genes, was performed and tested as a positive control for HPV 16 detection using PCR and real-time PCR. The results showed that the nucleotide and amino acid similarity of UI66 isolates varied, with the highest similarity observed in the partial L1 gene and E7 protein, respectively. The phylogeny of each gene from the UI66 isolate is variable. One clone carrying the recombinant plasmid pUI66F1-E6/E7 was obtained and can be used as a positive control for HPV 16 detection with E6 and E7 genes as targets.;HPV 16 is the most commonly detected genotype of HPV in cervical cancer. HPV 16 variants have been categorized based on their geographical distribution. Broer et al. have discovered HPV16 variants in Indonesia based on analysis of E6, E7 and L1. There has been no reports of other genes of HPV-16 Indonesian isolates. Methods for detection and genotyping of HPV DNA are the primary tools used to measure HPV infection and vaccine impact. Today, detection and genotyping HPV based on E6/E7 genes are being developed as E6 and E7 genes are the most conserved genes in cervical cancer patients. Standards and controls for the detection and genotyping accuracy are needed. WHO provides international standards in the form of a recombinant plasmid that contains the complete genome of HPV 16 and 18 from HPV genetic material abroad. Therefore in this research, cloning of fragments of HPV 16 E6/E7 Indonesian isolate have been done and used as a positive control for detection of HPV 16. The genome fragments of the HPV 16 UI66 isolates were amplified by PCR and sequenced then analyzed genetic variations of E6, E7, E1, E2, E4, E5, L2, partial L1 genes and LCR compared with HPV 16 reference. Cloning of fragment 1 contain E6/E7 genes have been done and tested as positive control of HPV 16 detection by real time PCR. The result showed the nucleotide and amino acid similarity of UI66 isolates varied with the highest value were partial L1 gene and E7 protein respectively. Phylogeny of each gene from isolate UI66 is variable. One clone was obtained carrying the recombinant plasmid pUI66F1-E6/E7 that can be used as a positive control for detection of HPV 16 which E6 and E7 genes as targets.;HPV 16 is the most commonly detected genotype of HPV in cervical cancer. HPV

16 variants have been categorized based on their geographical distribution. Broer

et al. have discovered HPV16 variants in Indonesia based on analysis of E6, E7

and L1. There has been no reports of other genes of HPV-16 Indonesian isolates.

Methods for detection and genotyping of HPV DNA are the primary tools used to

measure HPV infection and vaccine impact. Today, detection and genotyping

HPV based on E6/E7 genes are being developed as E6 and E7 genes are the most

conserved genes in cervical cancer patients. Standards and controls for the

detection and genotyping accuracy are needed. WHO provides international

standards in the form of a recombinant plasmid that contains the complete genome

of HPV 16 and 18 from HPV genetic material abroad. Therefore in this research,

cloning of fragments of HPV 16 E6/E7 Indonesian isolate have been done and

used as a positive control for detection of HPV 16. The genome fragments of the

HPV 16 UI66 isolates were amplified by PCR and sequenced then analyzed

genetic variations of E6, E7, E1, E2, E4, E5, L2, partial L1 genes and LCR

compared with HPV 16 reference. Cloning of fragment 1 contain E6/E7 genes

have been done and tested as positive control of HPV 16 detection by real time

PCR. The result showed the nucleotide and amino acid similarity of UI66

isolates varied with the highest value were partial L1 gene and E7 protein

respectively. Phylogeny of each gene from isolate UI66 is variable. One clone

was obtained carrying the recombinant plasmid pUI66F1-E6/E7 that can be used

as a positive control for detection of HPV 16 which E6 and E7 genes as targets., HPV 16 is the most commonly detected genotype of HPV in cervical cancer. HPV