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"Latar Belakang: Ada sekitar 50 spesies Nontuberculous mycobacteria (NTM) berpotensi patogen di manusia, yang menyebabkan infeksi tersering di paru. Pemeriksaan mikrobiologi berbasis molekuler diperlukan dalam mendiagnosis infeksi NTM paru. Oleh karena itu perlu dilakukan uji awal untuk deteksi mycobacteria dari spesimen klinis secara langsung dengan metode molekuler yaitu real-time PCR dan sekuensing DNA untuk identifikasi spesies mycobacteria serta mengaplikasikan pada kit PaxView® TB/NTM MPCR-ULFA. Tujuan: Melakukan optimasi berbasis molekuler untuk deteksi dan identifikasi Mycobacteria secara langsung pada sputum pasien terduga infeksi NTM paru. Metode: Studi dekskriptif dan eksperimental laboratorium dengan melakukan optimasi suhu penempelan, reaksi silang, ambang batas deteksi DNA, dan penerapan real-time PCR berbasis SYBR Green yang telahdioptimasi dan PaxView® TB/NTM MPCR-ULFA pada hasil sputum pasien terduga infeksi NTM paru. Hasil: Dua hasil positif dari 30 sampel sputum pada real-time PCR mycobacterium dan hasil sekuensing adalah Mycobacterium tuberculosis, menunjukkan adanya discordant hasil dengan real-time PCR MTB. Pada kit PaxView® TB/NTM MPCR-ULFA didapatkan 16 hasil positif MTB dan tidak ditemukan NTM. Kesimpulan: Terdapat discordance pada dua sampel hasil penerapan uji awal real-time PCR mycobacterium dengan sekuensing DNA, yang diduga NTM tetapi hasilnya M. tuberculosis. Perlunya dilakukan evaluasi lebih lanjut real-time PCR berbasis SYBR Green.

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Background: There are approximately fifty species of Nontuberculous mycobacteria (NTM) are potentially pathogenic in humans, the infection is most common in the lungs. Molecular-based microbiological examination is needed in diagnosing pulmonary NTM infection. Therefore, it is necessary to preliminary test the detection of mycobacteria from clinical specimens directly by molecular methods, namely real-time PCR and DNA sequencing to identify mycobacteria species and apply to the Pax-ULFA PaxView® TB/NTM kit. Aims: To perform Molecular-based optimization for the detection and identification of mycobacteria directly in sputum patient suspected of pulmonary NTM infection. Method: A descriptive and experimental laboratory study, to optimize the annealing temperature, determination of minimal detection of DNA, cross reaction of optimized real-time PCR based on SYBR- Green and applied sputum from patients suspected of NTM pulmonary infection to real-time PCR and PaxView® TB/NTM MPCR-ULFA. Results: Two positive results from 30 sputum samples on real-time PCR mycobacterium and sequencing results were MTB, the results discordant with real-time PCR MTB. In the PaxView® TB/NTM MPCR-ULFA, 16 positive MTB results were obatined and no NTM was found. Conclusion: There was discordance in two sample of real-time PCR mycobacterium spp. with DNA sequencing, which is thought to be NTM but the result is M. tuberculosis. The need for further evaluation of real-time PCR based Mikrobiologi Klinik on SYBR Green."

"Background: Currently available molecular method to detect HER2I655V polymorphism such as PCR-RFLP ishampered by costly experimental method and post-PCR treatment requirment that make this technique is not meetingfor high-throughput analysis purpose. In this study we developed a accurate, simple, low cost and rapid test to detectpolymorphism at HER2 gene using SBR Green I based-melting curve method. Methods: Two forward allele-specificprimers and one common reverse primer were used then these primers were tested to discriminate known genotypes ofgenomic templates (GG type or AA type) and genomic samples retrieved from breast cancer patients. Results: Meltingcurve analysis derived from SYBR Green I-based allele-specific PCR with defined primers concentration and annelingtemperature at 54.3 °C showed good discrimination level of Tm peaks in which GG genotype melted at 89 °C slightlyhigher than AA genotype which melted at 86 °C, while AG genotype harbored both of homozygous Tm characteristics.Conclusions: This preliminary result will be as basic for further large-scale typing of HER2I655V polymorphism."

"Resistensi Neisseria gonorrhoeae terhadap antibiotika merupakan masalah global di dunia. Sulitnya pertumbuhan N. gonorrhoeae di laboratorium menyebabkan uji kepekaan antibiotika sulit dilakukan secara reguler. Tujuan penelitian ini untuk mendeteksi N. gonorrhoeae dari spesimen endoserviks dan karakterisasi mutasi gen terkait resistensi terhadap sefiksim dan azitromisin sebagai antibiotika pilihan yang direkomendasikan WHO dan Kemenkes RI. Spesimen endoserviks dari wanita pekerja seks (WPS) dilakukan pewarnaan Gram, kultur, dan uji kepekaan antibiotika. Uji molekuler SYBR green real time PCR digunakan untuk mendeteksi N. gonorrhoeae, mutasi gen penA (Ala501Val/Pro, Gly545Ser) dan 23S rRNA (A2059G, C2611T). Resistensi 9 isolat N. gonorrhoeae terhadap sefiksim, levofloksasin, kanamisin sebesar 11,1%, 33,3%, 77,8% secara berurutan. Tidak ditemukan resistensi terhadap azitromisin dan seftriakson. Sedangkan resistensi terhadap penisilin, tetrasiklin, dan siprofloksasin ditemukan pada semua isolat. Uji SYBR green real time PCR berhasil mendeteksi N. gonorrhoeae dari spesimen endoserviks dan karakterisasi mutasi gen terkait resistensi terhadap sefiksim dan azitromisin. Dibandingkan pewarnaan Gram dan kultur, uji ini meningkatkan tingkat kepositifan sebesar 27% dan 15%. Tidak ditemukan mutasi pada gen penA dan 23S rRNA.

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Antimicrobial resistance in Neisseria gonorrhoeae is a global problem in the world. Due to N. gonorrhoeae is difficult to grow in the laboratory, antimicrobial susceptibility testing cannot be performed regularly. The aim of this study is to detect N. gonorrhoeae from endocervical specimens and to characterize gene mutations associated with cefixime and azithromycin resistance as the drugs of choice recommended by WHO and the Indonesian Ministry of Health. Endocervical specimens from female sex workers (FSW) were examined using Gram staining, culture, and susceptibility testing. Molecular SYBR green real-time PCR were used to detect N. gonorrhoeae and mutations in penA (Ala501Val/Pro, Gly545Ser) and 23S rRNA (A2059G, C2611T). Resistance of 9 isolates N. gonorrhoeae to cefixime, levofloxacin, kanamycin, were 11,1%, 33,3%, 77,8%, respectively. Resistance to azithromycin and ceftriaxone were not found. Whereas resistance to penicillin, tetracycline, and ciprofloxacin were found in all isolates. SYBR green real time PCR was successfully detect N. gonorrhoeae from endocervical specimens and characterize gene mutations associated with cefixime and azithromycin resistance. Compared to Gram and culture, this method could increase positivity rates as much as 27% and 15%. Mutation in penA and 23S rRNA were not found."

"Peningkatan kesadaran masyarakat muslim akan kehalalan produk farmasi menyebabkan kebutuhan sertifikasi halal produk farmasi terus meningkat. Metode pemeriksaan berbasis DNA telah disepakati sebagai salah satu pemeriksaan yang wajib dilakukan dalam pemeriksaan halal. qPCR berbasis SYBR Green merupakan metode pemeriksaan berbasis DNA yang memiliki kecepatan analisis yang lebih tinggi dibandingkan PCR konvensional dan lebih ekonomis dibandingkan qPCR berbasis probe. Multiplex PCR merupakan reaksi PCR yang menggabungkan beberapa primer dalam satu reaksi untuk mengamplifikasi beberapa gen secara sekaligus. Penggunaan primer universal telah dikembangkan untuk meningkatkan reprodusibilitas multiplex PCR berbasis SYBR Green, tetapi belum berhasil melakukan diskriminasi spesies hewan. Pada penelitian ini, primer forward universal yang didampingin dengan primer reverse spesifik untuk porcine, canine, dan murine berhasil dikembangkan. Selain itu, setiap primer menghasilkan amplicon dengan nilai Tm yang berbeda sehingga diskriminasi spesies hewan dapat dilakukan. Multiplex qPCR dari kombinasi primer tersebut ditemukan dapat mengamplifikasi ketiga gen secara sekaligus dengan variasi intra-assay dan variasi inter-assay sebesar 9,83% dan 11,53%. Multiplex qPCR yang dikembangkan dalam mendeteksi gen porcine dalam 6,81 pg/μL DNA total, gen murine dalam 22,88 pg/μL DNA total, dan gen canine dalam 88,06 pg/μL DNA total. Multiplex qPCR yang dikembangkan terbukti dapat mendeteksi sisa DNA yang terdapat pada produk farmasi maupun kosmetik.

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The rise of muslim awareness in halal pharmaceutical has caused an increase in demand for halal certified pharmaceutical. DNA based detection has been approved as a gold standard in halal examination. Intercalating dye-based qPCR is more economically available compared to available commercial kits which employs probe-based qPCR and also require less analysis time compared to conventional PCR. Multiplex qPCR could amplify more than one target by combining two primer set in one reaction. Universal primer have been developed to increase intercalating dye based Multiplex qPCR reproducibility. However, discrimination of animal species with universal primer have not been successful. In this study, universal forward primer was combined with a specific reverse primer for porcine, canine, and murine. These primers were found to be specific and were able to produce a different melting temperature, enabling animal species discrimination. Multiplex qPCR with these primers was repeatable with an intra assay variance and inter assay variance of 9,83% and 11,53%. The developed multiplex qPCR could detect porcine gene in 6,81 pg/μL DNA solution, murine gene in 22,88 pg/μL DNA solution, and canine gene in 88,06 pg/μL DNA solution. Moreover, the developed multiplex qPCR was proven to be able to detect DNA remnant in pharmaceutical and cosmeuticals."

"Virus dengue (DENV) adalah penyebab penyakit infeksi yang endemik di 100 negara di dunia. DENV dapat menyebabkan infeksi primer dan sekunder. Infeksi sekunder diperkirakan lebih berat dan akan menyebabkan menjadi Demam Berdarah Dengue (DBD) dan Sindrom Renjatan Dengue (SRD). Penatalaksanaan yang lebih dini dan tepat akan membantu mengurangi terjadinya kasus berat seperti DBD dan SRD. Teknik diagnostik yang dikembangkan belum ada yang dapat mendeteksi secara cepat dan tepat pada awal infeksi terutama untuk virus dengue strain Indonesia. Tujuan penelitian ini untuk mendapatkan kondisi yang optimal untuk deteksi DENV dan analisis validasi tehnik in house multiplek realtime Reverse Transcriptase-Polimerase Chain Reaction (rRT-PCR) sehingga dapat digunakan untuk deteksi dini dan cepat infeksi DENV. Rancangan penelitian ini adalah penelitian eksperimental laboratorium. Strain standar DENV diisolasi dengan kit Roche® dan spesimen diekstraksi dengan kit Qiagen®. Strain standar DENV digunakan untuk optimasi suhu annealing dan konsentrasi primer masing-masing serotipe DENV dengan metode in house multiplek rRT-PCR berbasis SYBR green. Sebagai pembanding digunakan RT-PCR konvensional dengan menggunakan primer di daerah C-PrM. Primer in house multiplek rRT-PCR didesain di daerah envelope pada masing masing serotipe. Analisis limit of detection (LOD) dilakukan dengan pengenceran titer virus 105, 104, 103, 102, 10 dan 1 FFU/ml (in house multiplek rRT-PCR) pada keempat serotipe. Hasil in house multiplek rRT-PCR dibandingkan dengan hasil RT-PCR konvensional Lanciotti pada pasien dengue yang telah masuk dalam kriteria inklusi dan eksklusi. Suhu annealing optimal didapatkan pada suhu 58oC sedangkan konsentrasi optimal masing-masing primer untuk in house multiplek rRT-PCR adalah 4 pmol. LOD RNA pada DENV-1, DENV-2, DENV-3, DENV-4 adalah 1 FFU/ml, 10 FFU/ml, 104 FFU/ml dan 1 FFU/ml. In house multiplek rRT-PCR dibandingkan RT-PCR konvensional mempunyai sensitivitas sebesar 100%, spesifisitas 94,2 %, nilai prediksi positif 85,7% dan nilai prediksi negatif 100 %. In house multiplek rRT-PCR berbasis SYBR green merupakan metode yang dini, cepat dan tepat untuk deteksi DENV pada awal infeksi dengan sensitivitas dan spesifisitas yang baik sebagai metode diagnostik infeksi dengue dimasa mendatang.

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Dengue virus (DENV) is an infectious disease that is endemic in 100 countries in the World. Dengue virus infections can cause primary and secondary. Secondary infection is estimated to be more severe DHF and SDD. Developed diagnostic technique that no one has been able to quickly and accurately detect the infection early, especially for the Indonesian strain of dengue virus. The purpose of this study is to obtain optimal conditions for the detection of dengue infection and analysis techniques in-house validation of multiplex real-time Reverse Transcriptase-Polymerase Chain Reaction (rRT-PCR) for the Indonesian strain of dengue virus.

Design of this study is an experimental research laboratory. Standard strains of dengue virus was isolated with a kit Roche® and the specimen was extracted with Qiagen® kit. Standard strains of dengue virus is used for optimization primer annealing temperature and the concentration primers of each serotype dengue virus by multiplex rRT-PCR method based on SYBR green. Primers for RT-PCR conventional based lanciotii et al while rRT-PCR primer was designed in the envelope gen at each serotype. Limit of detection (LOD) by diluting the virus titer 105, 104, 103, 102 FFU /ml performed by rRTPCR in all four serotypes.

The results of multiplex rRT-PCR compared with results of conventional RT-PCR in patients with dengue lanciotti superbly into the criteria inclusion and exclusion. Optimal annealing temperature results obtained at a temperature of 58oC and optimal primer concentration of 4 pmol of each primer for 25 ul total reaction. LOD RNA in DENV-1, DENV-2, DENV-3 and DENV-4 at titer of 1 FFU / ml, 10 FFU/ml, 104 FFU/ml and 1 FFU/ml. .In house multiplex rRT-PCR compared with RT-PCR has a sensitivity of 100%, specificity 95,2%, positive predictive value 85,7% and negative predictive value of 100%. In-house multiplex rRT-PCR with SYBR green-based research is a method that is rapid and precise detection of dengue virus in early infection with good sensitivity and specificity compared to RT-PCR as a diagnostic method in the future dengue infection."

"RT-qPCR (Real-Time Reverse-Transcription Polymerase Chain Reaction) berbasis SYBR Green, merupakan metode alternatif yang dapat digunakan untuk pendeteksian SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), selain berbasis TaqMan. Pada penelitian ini, primer yang dirancang menargetkan gen RdRP (Ribonucleic Acid/RNA-Dependent Ribonucleic Acid Polymerase) berdasarkan sekuens SARS-CoV-2 di Indonesia. Efisiensi dari metode yang dilakukan diketahui sebesar 97,82% dan limit of detection-nya adalah sampel dengan CT (cycle threshold) sebesar 41,25. Pada hasil uji spesifisitas, dua puluh sampel RNA positif SARS-CoV-2 terdeteksi positif dan delapan dari sepuluh sampel RNA negatif SARS-CoV-2 terdeteksi negatif. Dua sampel negatif yang terdeteksi positif karena terdeteksinya primer-dimer. Metode memenuhi kriteria presisi dengan hasil koefisien variasi pada intra-assay kurang dari 10% dan pada inter-assay kurang dari 15%. Sebagai langkah awal pengembangan deteksi kuantitatif, pada penelitian ini juga dilakukan percobaan penumbuhan transforman menggunakan sel kompeten One Shot® TOP10 dan One Shot® BL21(DE3) dengan plasmid kontrol DNA (deoxyribonucleic acid) pUC19 sebagai pemastian efisiensi sel kompeten yang dapat digunakan untuk penumbuhan kloning transforman gen RdRP SARS-CoV-2. Jumlah koloni bakteri yang berhasil tumbuh dari dua jenis sel kompeten tersebut tidak sesuai dengan ekspektasi panduan dari produsen. Selain itu, pada penelitian ini telah berhasil didapatkan fragmen gen target RdRP untuk kloning dengan PCR konvensional.

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SYBR Green-based RT-qPCR (Real-Time Reverse-Transcription Polymerase Chain Reaction) is an alternative method to detect SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), besides the TaqMan-based. In this study, the primers were designed to target the RdRP (Ribonucleic Acid/RNA-Dependent Ribonucleic Acid Polymerase) gene based on a SARS-CoV-2 sequence in Indonesia. The method's efficiency is known to be 97,82% and the limit of detection is a sample with a CT (cycle threshold) of 41,25. At the specificity test results, all twenty positive RNA samples of SARS-CoV-2 were detected as positive. Eight from ten negative RNA samples of SARS-CoV-2 were detected as negative, the remaining detected primer-dimer. The method meets the criteria of precision with the results of the coefficient of variation was less than 10% and 15% for intra-assay and inter-assay, respectively. As an initial step in developing quantitative detection, in this study, the efficiency of One Shot® TOP10 and One Shot® BL21(DE3) competent cells were confirmed using a DNA (deoxyribonucleic acid) control plasmid pUC19. Both types of competent cells do not meet the expectation based on the manufacturer. In addition, for cloning, the RdRP gene target fragment was successfully obtained by conventional PCR."

"ABSTRAKBenih bawal bintang memperlihatkan perubahan tingkah laku seperti kehilangan kemampuan berenang dan berkumpul di dasar kolam, diduga terinfeksi RSIVD. Real time PCR dengan SBYR green telah diaplikasikan secara luas untuk diagnosis penyakit. Kesederhanaan, sensitifitas, rentang deteksi yang dinamis, reproduktifitas, dan jaminan skrining dengan kecepatan waktu yang tinggi membuat real time PCR sesuai untuk mendeteksi virus (Niesters, 2002). Oleh karena itu dilakukan aplikasi metode real time PCR dengan SYBR green untuk mendeteksi RSIV pada benih bawal bintang. Penelitian ini menggunakan primer 1F dan 1R untuk skrining Megalocityvirus, grunt fin cell line untuk kultur RSIV, pengklonaan menggunakan vektor pGEM-T easy dan primer MCPspecR697-F4 dan MCP-specR888-R6 untuk deteksi RSIV dengan metode real time PCR menggunakan SYBR green. Karakterisasi dari CPE menunjukkan sel GF menjadi berbentuk bundar dan sel-sel GF terlihat berpendar pada inokulasi RSIV pada hari ke lima sampai ke tujuh. Kurva standar menghasilkan R2 0,99999, slope -2,41675 dan y-intercept 38,68938. Limit deteksi 10 salinan DNA. Spesimen klinis menunjukkan hasil positif pada jaringan hati, limpa dan ginjal. Jumlah salinan DNA paling banyak dari ekstraksi limpa yaitu: 6054 dan 4182 salinan DNA sedangkan pada organ ginjal sebanyak 72 dan 101 salinan DNA dan hati 1 dan 2 salinan DNA. Metode real time PCR menggunakan SYBR green berhasil diaplikasikan untuk mendeteksi RSIV pada benih ikan bawal bintang.

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ABSTRACTSnubnose pompano juvenile showed behavioral changes such as losed the ability to swim and congregated at the bottom of the pool, suspected of being infected RSIVD. Real time PCR with green SBYR has been widely applied to the diagnosis of the disease. Simplicity, sensitivity, dynamic range of detection, reproducibility, and the assurance screening with a high speed makes real time PCR according to detect viruses (Niesters, 2002). Therefore, it is done in real time application method with SYBR green PCR to detect RSIV on Snubnose pompano juvenile. This study using the primers 1F and 1R for screening Megalocityvirus, grunt fin cell line for RSIV culture, cloning using pGEM-T easy vector and primer MCPspecR697-F4 and MCP-specR888-R6 for RSIV detection by real-time PCR method using SYBR green.GF cells infected by RSIV showed round and flourescence as a result of CPE at day 5 to 7. Subnose pompano juvenile positive infected by RSIV at 191 bp. Standard curve equation R2: 0.99999, slope: -2.41675 and y-intercept: 38.68938. qPCR using primers MCP-specR674-F4 and MCP-specR888 R6 primer assay showed detection limit of 10 copies of the. Liver, spleen and kidneys of Subnose Dart juvenile were infected by RSIV, positively. The highest of copy number of DNA were shown in the spleen (6054 and 4182 copies DNA, respectively), while in kidney were 101 and 72 copies DNA respectively. The lowest copy number DNA were shown in the liver (1 to 2 copies DNA, respectively). SYBR green quantitative PCR method can be applied to detect RSIV on Subnose pompano juvenile."