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Abstrak :
Pandemi virus corona SARS-CoV-2 di seluruh dunia telah menyebabkan besarnya populasi manusia yang terinfeksi COVID-19. Penyebaran virus yang massive membutuhkan alat diagnostik yang cepat, sehingga keputusan terkait kebijakan kesehatan, pembatasan publik, dan keputusan karantina dapat diambil dengan tepat. Polymerase chain reaction (PCR) adalah standar emas dalam pengujian asam nukleat yang mendeteksi viral ribonucleic acid (RNA). Meskipun real-time RT- PCR sensitif dan reliabel, namun prosesnya memakan waktu yang cukup lama sehingga tidak cukup untuk menjawab kebutuhan diagnosis di masa pandemi global COVID-19. Amplifikasi isotermal merupakan salah satu metode yang dapat digunakan sebagai alternatif PCR. Metode amplifikasi isotermal yang paling banyak diterapkan adalah loop-mediated isothermal amplification (LAMP). Pada penelitian ini dilakukan perancangan prototipe berbasis pemanas poliamida yang dapat mempermudah proses reaksi LAMP. Sistem ini dapat menghasilkan suhu konstan 60˚C-65˚C dalam 30 menit dengan nilai error rata-rata pengukuran sensor suhu sebesar 1.75% dengan akurasi 98.25%. Pada pengujian perbandingan nilai suhu pada sensor dengan suhu dalam tube terdapat error rata-rata sebesar 1.75% dengan akurasi 95.45%. Kuantifikasi intensitas fluoresens juga telah berhasil dilakukan dengan tingkat zat fluorosensi yang berbeda-beda dengan membaca nilai keabuan minimal 102,76 ......The SARS-CoV-2 coronavirus pandemic worldwide has caused a large number of people to be infected with COVID-19. The massive spread of the virus requires rapid diagnostic tools, so that appropriate health, public and policy decisions can be made. Polymerase chain reaction (PCR) is the gold standard in nucleic acid testing that detects viral ribonucleic acid (RNA). Although real-time RT-PCR is sensitive and reliable, the process takes a long time so it is not sufficient to answer the need for diagnosis during the global COVID-19 pandemic. Isothermal amplification is one method that can be used as an alternative PCR. The most widely applied isothermal amplification method is loop-mediated isothermal amplification (LAMP). In this study, a prototype based on a polyimide heater was designed that could facilitate the LAMP reaction process. This system can produce a constant temperature of 60˚C-65˚C in 30 minutes with an average error value of 1.75% of temperature sensor measurements with an accuracy of 98.25%. In testing the comparison of the temperature value on the sensor with the temperature in the tube, there is an average error of 1.75% with an accuracy of 95.45%. Fluorescence intensity quantification has also been successfully carried out with different levels of fluorescence intensity and can read a minimum gray value of 102,76

Abstrak :
Amplifikasi isotermal merupakan metode yang popular pada saat ini sebagai alternatif PCR (polymerase chain reaction) untuk melakukan amplifikasi asam nukleat. Tidak seperti PCR yang memerlukan perubahan suhu siklik, amplifikasi dapat dilakukan pada suhu konstan dalam metode amplifikasi isotermal. Salah satu metode amplifikasi isotermal yang sangat banyak diaplikasikan adalah loop-mediated isothermal amplification (LAMP). Sejak pertama kalinya ditemukan pada tahun 2000, LAMP dimanfaatkan secara luas untuk deteksi berbagai jenis patogen. Dalam penelitian ini, dirancang suatu prototipe yang dapat memfasilitasi reaksi LAMP. Prototipe terdiri dari dua sistem: sistem pemanas yang berfungsi untuk menyediakan suhu konstan 60oC terhadap sampel untuk aplikasi LAMP dan sistem deteksi optik berbasis fluoresens yang digunakan untuk melakukan kuantifikasi intensitas fluoresens dalam 6 tabung sampel 0,5mL dengan kadar fluorescence agent 0,5mL, 0,4mL, 0,3mL, 0,2mL, 0,1mL, dan 0mL. Agar panas dapat disalurkan sampel dengan baik, dirancang sebuah blok panas (heat block). Selain itu, sebuah housing prototipe juga dirancang untuk menyokong komponen dan mendukung proses deteksi fluoresens. Berdasarkan hasil penelitian ini, sistem pemanas sederhana menggunakan kontrol PID dan blok panas yang dirancang mampu mempertahankan suhu pada setpoint 60oC dan 65oC, sehingga dapat digunakan untuk aplikasi LAMP. Selain itu, sistem optik yang dirancang mampu memfasilitasi proses deteksi optik berbasis fluoresens. Proses kuantifikasi intensitas fluoresens menunjukkan bahwa intensitas emisi fluoresens proporsional terhadap kadar fluorescence agent dalam sampel, dimana sampel dengan total volume 0,5mL pada kadar fluorescence agent 0,5mL, 0,4mL, 0,3mL, 0,2mL, maupun 0,1mL dapat menghasilkan emisi cahaya dengan nilai keabuan minimal 121,179. ......Isothermal amplification is a popular method today as an alternative to PCR (polymerase chain reaction) for nucleic acid amplification. Unlike PCR which requires cyclic temperature changes, amplification can be carried out at a constant temperature in the isothermal amplification method. One of the most widely applied isothermal amplification methods is loop-mediated isothermal amplification (LAMP). Since it was first discovered in 2000, LAMP has been widely used to detect various types of pathogens. In this research, a prototype is designed that can facilitate the LAMP reaction. The prototype consists of two systems: a heating system which serves to provide a constant temperature of 60oC to the sample for the LAMP application and a fluorescence-based optical detection system which is used to quantify the fluorescence intensity in 6 sample tubes of 0.5mL with fluorescence agent content of 0.5mL, 0.4mL, 0.3mL, 0.2mL, 0.1mL, and 0mL. In order for heat to be transferred to the sample properly, a heat block is designed. In addition, a prototype housing is also designed to support components and support the fluorescence detection process. Based on the results of this study, the designed simple heating system with PID control and heating block are able to maintain a temperature at a setpoint of 60oC dan 65oC, so it can be used for LAMP applications. Moreover, the designed optical system is able to facilitate fluorescence-based optical detection process. The fluorescence intensity quantification process showed that fluorescent emission intensity is proportional to the content of fluorescence agent in the sample, where the sample with total volume of 0.5mL in which the fluorescence agent content is 0.5mL, 0.4mL, 0.3mL, 0.2mL, and 0.1mL can generate light emission with gray value of minimal 121.179.

Abstrak :
Penelitian ini merupakan usaha untuk mengembangkan suatu metode baru dalam mendeteksi HIV. Teknik deteksi yang biasa digunakan adalah RT-PCR dari sampel berupa RNA, Tujuan dari penelitian ini adalah untuk mengaplikasikan pengembangan metode amplifikasi DNA, LAMP (Loop-Mediated Isothermal Amplification), untuk menggantikan RT-PCR, karena metode LAMP ini dinilai lebih spesifik, sensitif, dan efisien. Reaksi LAMP telah dilakukan pada isolat RNA yang sebelumnya telah di-reverse transcription (RT) dan dikonfirmasi dengan PCR. Reaksi tersebut menggunakan enzim Bs/ DNA polimerase dan reaksinya berlangsung pada suhu 65°C. Hasil reaksi tersebut telah dikonfirmasi dengan elektroforesis dan menunjukkan ketiadaan pita hasil amplifikasi yang diharapkan. Argumentasi yang paling memungkinkan dari hasil reaksi ini antara lain, adalah kondisi kemurnian sampel, kondisi reaksi yang tidak optimal untuk reaksi LAMP, dan rancangan primer. Reaksi LAMP juga akan dilakukan pada bagian dari sekuens gen gag yang terletak pada nukleotida nomor 905-1081 dan telah diklon pada vektor pGEM-T serta telah dikonfirmasi dengan sekuensing DNA. Hasil sekuensing menunjukkan sekuens yang sama dengan data gene bank dengan ukuran yang sesuai dengan ukuran target primer komersial, yaitu sebesar 155 pb dan akan digunakan sebagai template untuk reaksi LAMP. Kesimpulan penelitian ini adalah metode LAMP helum berhasil dikembangkan untuk deteksi HIV. Rancangan primer dan kondisi reaksi adalah hal-hal yang penting dalam metode LAMP dan harus ditingkatkan untuk keberhasilan reaksi ini.

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This study was attempted to develop a new method for HIV detection. The technique that is usually used is RT-PCR for RNA detection. This research aims to apply the recently developed DNA amplification method, LAMP (Loop-Mediated Isothermal Amplification), instead of RT-PCR as this method is more specific, sensitive, and efficient. The LAMP reaction is done on RNA isolates that have been confirmed by PCR to contain HIV RNA. This reaction has been performed at 65°C using Bst DNA polymerase after doing reverse transcription. The result showed that LAMP on RNA isolates did not result in amplification as confirmed by electrophoresis. Most probable reasons for these results are the impurity of the sample, the conditions of the reactions that are not optimal for the LAMP reaction, and the design of the primer. LAMP was also performed on a part of a gag genes sequences on nucleotides number 905-1081 that has been cloned onto a pGEM-T vector and then sequenced for confirmation. The result of DNA sequence showed the same sequence as reported in Gene Bank data, with a size similar to a commercial primary target, i.e. 155 bp and will consequently be used as a template for the LAMP reaction. The conclusions of this study are the LAMP method has not developed yet for HIV detection. The design of the primer and the conditions of the reactions in the LAMP method are the important things for the successful of this reaction, need to be improved.

Abstrak :
Pandemi virus SARS-CoV-2 yang terjadi telah membuat kebutuhan untuk pemeriksaan massal deteksi virus menjadi meningkat. Saat ini pemeriksaan gold standard untuk mendeteksi virus SARS-CoV-2 adalah dengan metode reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) yang berbiaya mahal. Oleh karena itu dibutuhkan metode pemeriksaan alternatif yang lebih murah, cepat, dan mudah digunakan sebagai sistem deteksi virus. Metode Loop Mediated Isothermal Amplification LAMP) dapat melakukan reaksi amplifikasi nukleotida pada suhu isothermaltanpa perlu mesin thermal cycler dan dapat diamati langsung dengan penambahan zat pewarna. Metode deteksi virus berbasis LAMP yang dikembangkan menggunakan desain primer yang menarget gen S dan ORF1ab ditambah pewarna Calcein-Mangan dan senyawa tambahan Guanidine Hydrochloride (GuHCl). Sistem deteksi dilakukan optimasi konsentrasi komponen reaksi, suhu dan template yang menggunakan plasmid DNA kontrol. Optimasi didapatkan dengan konsentrasi reaksi FIP/BIP 0,4 µM, F3/B3 0,2 µM, Loop F/P 0,4 µM, Calcein-Mangan 12 µM, dan GuHCl 40 mM. Sistem LAMP yang dikembangkan dapat mendeteksi sekuens gen target pada DNA kontrol hingga 1 copy number dalam waktu sekitar 1 jam pada inkubasi suhu 55 ºC. Meski begitu, sistem LAMP yang dikembangkan belum mapu mengamplifikasi RNA virus SARS-CoV-2 hasil ekstraksi sampel swab pasien. Hal ini disebabkan oleh enzim Bst 3.0 yang dipakai dalam sistem LAMP tidak memiliki kemampuan reverse transcriptase yang diharapkan. ......The SARS-CoV-2 virus pandemic has made the need for mass screening of virus detection increased. Currently, the gold standard  test to detect SARS-CoV-2 virus is reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method that costly. Therefore, an alternative method that is cheaper, faster, and easier to use as a virus detection system is needed. The Loop Mediated Isothermal Amplification (LAMP) method can perform nucleotide amplification reactions at isothermal temperatures  without the need of thermal cycler  machine and can be observed directly with the addition of coloring agents. The LAMP-based virus detection method development use primers design targeting the S and ORF1ab genes with Calcein-Manganese dyes and the additive compound Guanidine Hydrochloride (GuHCl). The detection system performed optimization of the concentration of reaction components, temperature and template using plasmid DNA control. Optimization was obtained with reaction concentration of FIP/BIP 0.4 μM, F3/B3 0.2 μM, Loop F/P 0.4 μM, Calcein-Manganese 12 μM, and GuHCl 40 mM. The developed LAMP system can detect target gene sequences in control DNA up to  1 copy number in about 1 hour at 55 ºC incubation temperature. Even so, the LAMP system developed still unable to amplify the RNA of the SARS-CoV-2 virus from the extraction of patient swab samples. This issue occurred due to the Bst 3.0 enzyme that used in the reaction did not have reverse transcriptase activity as expected.

Abstrak :
Pendeteksian dini penyakit adalah hal yang paling penting untuk melawan suatu wabah penyakit. Akan tetapi hal tersebut menjadi masalah untuk wilayah ataupun daerah yang memiliki akses terbatas terhadap peralatan medis. Loop-Mediated Isothermal Amplification Polymerase Chain Reaction atau LAMP-PCR adalah suatu alat yang mampu mengamplifikasi suatu DNA, sehingga DNA tersebut akan mudah untuk dideteksi. Salah satu komponen yang diperlukan untuk alat LAMP PCR adalah modul pemanas, karena LAMP-PCR bekerja pada suhu yang tinggi yaitu sekitar 65oC. Panas yang dibutuhkan pada alat PCR dapat dihasikan dengan menggunakan suatu electrode dengan bahan campuran silver-polyvinyl alcohol (PVA) yang dialiri oleh listrik. Elektrode silver-pva tersebut akan mengalami fenomena resistive heating sehingga menghasilkan panas. Pada penelitian ini, dilakukan karakterisasi pada pemanas dengan bahan campuran silver-pva. Karakterisasi yang diteliti adalah electrical properties, mechanical properties, dan heat properties. Dari penelitian ini, didapat nilai electrical dan mechanical properties dari elektrode pemanas silver-pva meningkat sebanding dengan semakin tinggi konsentrasi PVA. Diperoleh juga temperature yang dapat dihasilkan oleh electrode pemanas silver-PVA ini adalah sebesar 102oC. ......Early detection of disease is the most important thing to fight against a disease outbreak. However, this is a problem in regions that have limited access to medical equipment. Loop-Mediated Isothermal Amplification Polymerase Chain Reaction or LAMP-PCR is a tool that can amplify a DNA, so that DNA will be easy to detect. One of the components needed for the LAMP PCR device is the heating module, because the LAMP-PCR works at high temperatures. The heat needed on the PCR can be generated by using an electrode with a silver-polivinyl alcohol (PVA) mixture which is electrically flowed. The silver-PVA electrode will experience a resistive heating phenomenon so that it will generate heat. In this study, characterization of heaters with a silver-pva mixture was carried out. The characters examined in this study are electrical properties, mechanical properties, and heat properties. From this study, the electrical and mechanical properties of the silver-pva heating electrode increased in proportion to the higher PVA concentration. The temperature that can be produced by the silver-PVA heating electrode is 102oC.

Abstrak :
ABSTRAK
Development of the Rapid Test Kit for the Identification of Campylobacter spp. Based on Loop-mediated Isothermal Amplification (LAMP) in Combination with a Lateral Flow Dipstick (LFD) and Gold Nano-DNA Probe (AuNPs) Dueantem Thongphueak, Kosum Chansiri Center of Excellence in Biosensors, Srinakharinwirot University, Bangkok 10110, Thailand. Thayat Sriyapai Faculty of Environmental Culture and Ecotourism, Srinakharinwirot University, Bangkok 10110, Thailand. Supatra Areekit, Somchai Santiwatanakul, Piyada Wangroongsarb ABSTRAK
The detection of Campylobacter spp. in meat products was developed by using loop-mediated isothermal amplification (LAMP) combined with DNA-based bioassay methods, including a lateral-flow dipstick (LFD) and gold nano-DNA probe (AuNPs) assay. The LAMP primers were designed from the conserved nucleotide regions of Campylobacter spp. The analytical sensitivity of the LAMP-LFD and LAMP-AuNPs analysis was 360 fg/μl. The analytical specificity of LAMP-based assays showed no cross-reactions to Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Serratia marcescens, Vibrio parahaemolyticus, Vibrio cholerae, Klebsiella oxytoca and Citrobacter diversus.The sensitivity, specificity and accuracy of both LAMP-LFD and LAMP-AuNPs for the detection of pre-enrichment cultures from raw chicken meat samples were 100%, 95% and 96.67%, respectively. Since the processing time of LAMP-based assays is 60-90 minutes, it is applicable as a point-of-care screening test for food safety and as a process control of Campylobacter spp. contamination.

Abstrak :
ABSTRACT
Hepatitis B virus (HBV) infects hepatocytes and causes acute and chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma (HCC) in both animals and humans. Early detection of HBV infection assists in monitoring the patient's response to anti-HBV therapy, blood donation screening, and disease management, control and eradication. This research focused on development of LAMP assay combined with lateral flow dipstick (LFD), gold nanoparticle (AuNPs) and real-time turbidimetry for screening of the hepatitis B virus. Analytical sensitivity, analytical specificity, diagnostic sensitivity, diagnostic specificity, accuracy and predictive value of each technique were determined and compared to conventional PCR and real-time PCR (gold standard method). The analytical sensitivity of LAMP-LFD and LAMP-AuNPs was 1.24x101 copies /mL, LAMP-real-time turbidimetry was 1.24x102 copies/mL, while that of conventional PCR was 1.24x104 copies/mL. Examination of the analytical specificity of all LAMP-based combinations and conventional PCR showed no cross-reactivity with HCV or human plasma. Upon exploration of one hundred unknown samples, in comparison to real-time PCR, the diagnostic sensitivity and specificity of LAMP-based assays were 100% and 90%, respectively. The accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the LAMP-based assays were 98%, 97.56%, and 100%, respectively. While that of conventional PCR were 60%, 100%, 68%, 100% and 38% of diagnostic sensitivity, diagnostic specificity, accuracy, PPV and NPV, respectively. LAMP-based assays need to be simplified in terms of achieving single-step diagnosis using one master mix solution that is suitable for a point-of-care diagnostic test.