Putu Irma Handayani

IN SILICO PRIMER DESIGN AND ANNEALING TEMPERATURE OPTIMIZATION TO AMPLIFY THE FRAGMENT OF gyrB GENE Mycobacterium tuberculosis ISOLATE P010 USING POLYMERASE CHAIN REACTION

Introduction

in silico primer design and annealing temperature optimization to amplify the fragment of gyrb gene mycobacterium tuberculosis isolate p010 using polymerase chain reaction. Optimize PCR amplification of Mycobacterium tuberculosis gyrB gene fragments. In silico primer design and annealing temperature optimization (56°C) reveal best primers for detecting XDR-TB mutations.

Abstract

One of the factors causing XDR-TB is due to mutations in the Mycobacterium tuberculosis gene, one of them is in the gyrB gene. Amplification of gyrB gene fragments from Mycobacterium tuberculosis DNA using Polymerase Chain Reaction (PCR) method. The amplification process by the PCR method requires a pair of primers (forward and reverse) to limit the area to be amplified. The current study aims to obtain the best primer pair generated by in silico design using Clone Manager Suite 6 program while simultaneously optimizing the annealing temperature to amplify the fragment of gyrB Mycobacterium tuberculosis. The template used in designing the primer is the sequence of gyrB Mycobacterium tuberculosis H37Rv isolate obtained from NCBI database of genbank code AL123456.3. The current study obtained a pair of primer which respectively had 19 oligonucleotide length and the best annealing temperature of 56ºC. The primer is be able to do in silico amplification of the fragment of gyrB Mycobacterium tuberculosis gene isolate P010 in the nucleotide area range from 1271-1755 bp with 485 bp fragment length.

Review

This manuscript addresses a highly relevant topic concerning the development of molecular tools for detecting *Mycobacterium tuberculosis*, particularly in the context of XDR-TB and mutations within the *gyrB* gene. The study's primary objective was to perform *in silico* primer design and annealing temperature optimization for the amplification of a *gyrB* gene fragment. Such *in silico* work is a crucial preliminary step in establishing robust PCR assays, which are vital for diagnostic purposes and research into drug resistance mechanisms, thereby contributing to efforts against multidrug-resistant tuberculosis. The authors utilized standard bioinformatics tools, specifically Clone Manager Suite 6, and a well-established reference sequence (H37Rv gyrB gene from NCBI GenBank) to achieve their aims. They successfully identified a pair of 19-oligonucleotide primers and determined an optimal annealing temperature of 56°C. The precise details provided, such as the target nucleotide range (1271-1755 bp) and the resulting 485 bp fragment length for *M. tuberculosis* isolate P010, indicate a thorough *in silico* characterization. These findings lay a clear theoretical groundwork for developing a specific PCR assay. However, a significant limitation of the presented work, as outlined in the abstract, is the exclusive reliance on *in silico* data. While the *in silico* design is well-executed, the absence of experimental validation means that the practical utility and robustness of the designed primers and optimized annealing temperature remain unconfirmed. For a study focused on PCR methodology, demonstrating successful amplification, specificity, and efficiency in a wet-lab setting using actual *M. tuberculosis* DNA (especially isolate P010) is imperative. Future work must prioritize experimental PCR to validate these predictions, confirm the absence of non-specific amplification, and assess the primers' performance on clinical isolates, thereby transforming this theoretical design into a practical and reliable diagnostic or research tool.

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