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All Journal Science and Technology Indonesia Jurnal Bioteknologi & Biosains Indonesia (JBBI) Jurnal Bioteknologi & Biosains Indonesia
Zahro, Aurora Fatimatuz
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Environmental Science Health Professions Materials Science & Nanotechnology Physics

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Advancements in Real-Time PCR Technologies: A Comprehensive Review of Probe-Based and Non-Probe-Based Assays for Molecular Diagnostics Malau, Jekmal; Zahro, Aurora Fatimatuz; Zahra, Aliya Azkia; Kasasiah, Ahsanal; Meilani, Nanda Diva; Damara, Dandy Satria; Lestari, Agatha Nabilla; Saryono; Wahyono, Daniel Joko
Science and Technology Indonesia Vol. 10 No. 3 (2025): July
Publisher : Research Center of Inorganic Materials and Coordination Complexes, FMIPA Universitas Sriwijaya

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.26554/sti.2025.10.3.660-677

Abstract

The decision between probe-based and non-probe-based qPCR assays is crucial, influenced by diagnostic goals and sample characteristics. This review provides an in-depth evaluation of these two assay types, analyzing their principles, strengths, drawbacks, and applications. A thorough review of the literature, primarily sourced from PubMed, was undertaken to explore prominent assay systems, including TaqMan, KASP, rhAmp, HRM, and SYBR Green. Probe-based qPCR assays, exemplified by TaqMan and rhAmp, are distinguished by their high specificity, aptitude for multiplex analysis, and reduced risk of false positives, making them highly suitable for SNP genotyping and pathogen detection. However, their elevated costs and intricate design requirements remain significant challenges. Conversely, non-probe-based assays, such as SYBR Green and HRM, present cost-effective alternatives with straightforward designs. HRM, in particular, is effective in identifying genetic variations like SNPs with remarkable sensitivity. Nonetheless, these methods are susceptible to non-specific amplifications, requiring careful optimization to maintain reliability. The selection of a suitable qPCR assay depends on various factors, including precision, affordability, and multiplexing capabilities, with applications spanning infectious disease detection and genetic disorder analysis. This review emphasizes the indispensable role of qPCR in molecular diagnostics while showcasing recent technological advances that aim to mitigate existing constraints and enhance diagnostic precision and accessibility.
DEVELOPMENT OF PLASMID-BASED FOR EXTERNAL CONTROL MATERIALS OF CYP2D6*10 (rs1065852) GENE PCR-BASED DETECTION Malau, Jekmal; Zahra, Aliya Azkia; Kasasiah, Ahsanal; Rahmasari, Ratika; Raekiansyah, Muhareva; Rohmah, Siti; Meilani, Nanda Diva; Septi, Annisa Frastica; Zahro, Aurora Fatimatuz; Annajla, Fathina; Hermosaningtyas, Anastasia Aliesa; Hilmi, Indah Laily
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 10 No. 2 (2023)
Publisher : BRIN - Badan Riset dan Inovasi Nasional

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.55981/jbbi.2023.2557

Abstract

Reliable clinical diagnosis of Single Nucleotide Polymorphisms (SNPs) is necessary for personalizing tamoxifen medication according to CYP2D6*10 genetic variations. Our research aimed to create a recombinant plasmid for external control material with a molecular size of 3812 bp. The recombinant plasmid was achieved by cloning an 838 bp gene insert of CYP2D6*10 rs1065852 into a 2974 bp pJET1.2 plasmid into Escherichia coli DH10B and selection on ampicillin agar medium. Isolated E. coli recombinants provided the plasmid molecules for analysis. Bi-directional sequencing and Real-Time PCR confirmed the presence of wild-type and mutant rs1065852 DNA fragments in the plasmid, namely homozygote CC and TT. The conclusion is that we have successfully introduced a novel recombinant plasmid developed by cloning the SNP rs1065852, which carries the 100C>T mutation, using pJET 1.2/blunt system, which could significantly enhance the accuracy of clinical SNP diagnostics for personalized medicine in breast cancer treatment.
DEVELOPMENT OF PLASMID-BASED FOR EXTERNAL CONTROL MATERIALS OF CYP2D6*10 (rs1065852) GENE PCR-BASED DETECTION Malau, Jekmal; Zahra, Aliya Azkia; Kasasiah, Ahsanal; Rahmasari, Ratika; Raekiansyah, Muhareva; Rohmah, Siti; Meilani, Nanda Diva; Septi, Annisa Frastica; Zahro, Aurora Fatimatuz; Annajla, Fathina; Hermosaningtyas, Anastasia Aliesa; Hilmi, Indah Laily
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 10 No. 2 (2023)
Publisher : BRIN - Badan Riset dan Inovasi Nasional

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.55981/jbbi.2023.2557

Abstract

Reliable clinical diagnosis of Single Nucleotide Polymorphisms (SNPs) is necessary for personalizing tamoxifen medication according to CYP2D6*10 genetic variations. Our research aimed to create a recombinant plasmid for external control material with a molecular size of 3812 bp. The recombinant plasmid was achieved by cloning an 838 bp gene insert of CYP2D6*10 rs1065852 into a 2974 bp pJET1.2 plasmid into Escherichia coli DH10B and selection on ampicillin agar medium. Isolated E. coli recombinants provided the plasmid molecules for analysis. Bi-directional sequencing and Real-Time PCR confirmed the presence of wild-type and mutant rs1065852 DNA fragments in the plasmid, namely homozygote CC and TT. The conclusion is that we have successfully introduced a novel recombinant plasmid developed by cloning the SNP rs1065852, which carries the 100C>T mutation, using pJET 1.2/blunt system, which could significantly enhance the accuracy of clinical SNP diagnostics for personalized medicine in breast cancer treatment.
Co-Authors Ahsanal Kasasiah Annajla, Fathina Damara, Dandy Satria Daniel Joko Wahyono Hermosaningtyas, Anastasia Aliesa Indah Laily Hilmi Jekmal Malau Lestari, Agatha Nabilla Meilani, Nanda Diva Muhareva Raekiansyah Rahmasari, Ratika Saryono Septi, Annisa Frastica Siti Rohmah Zahra, Aliya Azkia