scholarly journals Deteksi Vibrio harveyi dengan Metode Amplifikasi DNA pada Gen toxR

2020 ◽  
Vol 1 (1) ◽  
pp. 29-37
Author(s):  
Calvin Wijaya Johan ◽  
Sulistyo Emantoko Dwi Putra ◽  
Ernest Suryadjaja
Keyword(s):  
False Positive ◽  
Vibrio Harveyi ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain ◽  
Toxr Gene ◽  
Positive Results

Abstrak -Infeksi hewan akuakultur oleh Vibrio harveyi dapat menyebabkan kematian serta kerugian ekonomi. Kemampuan untuk mendeteksi bakteri tersebut secara dini dapat mencegah penyebarannya dalam akuakultur. Penelitian ini bertujuan untuk mengembangkan metode untuk mendeteksi Vibrio harveyi melalui amplifikasi gen toxR. Amplifikasi DNA dilakukan dengan dua metode, yakni amplifikasi isotermal termediasi loop (loop-mediated isothermal amplification, LAMP) dan reaksi berantai polimerase (PCR). Amplifikasi menggunakan metode LAMP menunjukan perlu dilakukan optimasi protokol maupun desain primer untuk mencegah perolehan hasil false positive. Amplifikasi menggunakan metode PCR menghasilkan produk berukuran 229 pasang basa yang spesifik pada Vibrio harveyi dengan batas deteksi hingga 0,526 ng.µL-1 (setara 2,09 × 106 CFU.mL-1). Kata kunci: Akuakultur, LAMP, PCR, toxR, Vibrio harveyi Abstract -Vibrio harveyi infection in aquacultures may cause death and economical loss. Rapid detection of this bacteria may prevent its dispersal in aquacultures. The goal of this research was to develop method in detection of Vibrio harveyi via amplification of toxR gene. DNA amplification was carried out with two methods, loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Amplification with LAMP suggest optimization of either protocol or primer design was needed to prevents false positive results. Amplification with PCR yields 229 bp-length product specific to Vibrio harveyi with detection limit up to 0.526 ng.µL-1 (equals to 2.09 × 106 CFU.mL-1). Keywords: Aquaculture, LAMP, PCR, toxR, Vibrio harveyi

scholarly journals LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF C. BURNETII IN BULGARIA

2021 ◽  
Vol 19 (2) ◽  
pp. 147-151
Author(s):  
M. Kunchev ◽  
V. Belcheva ◽  
E. Grigorov
Keyword(s):  
Polymerase Chain Reaction ◽  
Sensitivity And Specificity ◽  
Q Fever ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Retrospective Diagnosis ◽  
Loop Mediated Isothermal Amplification ◽  
The World ◽  
Polymerase Chain

Q fever, which is caused by Coxiella burnetii, a small, pleomorphic intracellular bacterium, is the most widespread zoonosis in the world. The chronic form of the disease can lead to disability and death. Rapid diagnosis of Q fever is needed in order that effective treatment can be initiated. The conventional retrospective diagnosis of Q fever, based on serology, is useless for the treatment of afflicted patients. Thus, molecular methods have been created to close the diagnostic gap between the onset of the disease and the presence of specific antibodies in serum. A polymerase chain reaction is a suitable and reliable method with high sensitivity and specificity, but it requires expensive equipment and post-amplification protocol. Loop-mediated isothermal amplification (LAMP) is an isothermal technique, conducted at constant temperature that can amplify a negligible amount of DNA to more than 109 copies within one hour, using special primers and polymerase. We have tested the sensitivity and specificity of LAMP in the detection of C. burnetii. The mean positive rate of LAMP and polymerase chain reaction in patients was 100% and 74%, respectively. LAMP reacted negatively with non-C. burnetii pathogens and non-infected blood samples. We conclude that LAMP is a sensitive and specific technique for the detection of C. burnetii and has advantages over serological methods and PCR that make it attractive for diagnosing Q fever in countries around the world.

scholarly journals Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research

Animals ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 76
Author(s):  
Faiz Padzil ◽  
Abdul Razak Mariatulqabtiah ◽  
Wen Siang Tan ◽  
Kok Lian Ho ◽  
Nurulfiza Mat Isa ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Isothermal Amplification ◽  
Screening Methods ◽  
Diagnostic Strategy ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain

Over the years, development of molecular diagnostics has evolved significantly in the detection of pathogens within humans and their surroundings. Researchers have discovered new species and strains of viruses, while mitigating the viral infections that occur, owing to the accessibility of nucleic acid screening methods such as polymerase chain reaction (PCR), qualitative (real-time) polymerase chain reaction (qPCR) and reverse-transcription qPCR (RT-qPCR). While such molecular detection methods are widely utilized as the benchmark, the invention of isothermal amplifications has also emerged as a reliable tool to improvise on-field diagnosis without dependence on thermocyclers. Among the established isothermal amplification technologies are loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement activity (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and rolling circle amplification (RCA). This review highlights the past research on and future prospects of LAMP, its principles and applications as a promising point-of-care diagnostic method against avian viruses.

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