Study guide
8.4 Isothermal & Target Multiplex Amplification
ASCP Molecular Biology Technologist MB
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1 Introduction & Exam Strategy
1.1 ASCP MB Exam Structure, Eligibility Routes & Policies 1.2 Computer Adaptive Testing (CAT) & Scoring Mechanics 1.3 Blueprint Navigation & Preparation Strategy
2 Nucleic Acid Chemistry & Molecular Structure
2.1 DNA Structure, Nitrogenous Bases & Thermodynamic Stability 2.2 RNA Types, Secondary Structure & Chemical Lability 2.3 Chromatin Organization, Histone Modifications & DNA Methylation
3 Central Dogma, Genome Organization & Cytogenetics
3.1 DNA Replication Machinery & Enzymology 3.2 Transcription, Translation & Genetic Code 3.3 Mutations, Polymorphisms & Chromosomal Aberrations
4 Biochemical Reagents & Enzymes
4.1 DNA Polymerases, Reverse Transcriptases & Proofreading 4.2 Restriction Endonucleases, Ligases & Nucleases 4.3 Reaction Buffers, Cations & Master Mix Additives
5 Nucleic Acid Isolation & Purification
5.1 Organic & Inorganic Extraction Chemistries 5.2 Solid-Phase Silica Columns & Magnetic Bead Isolation 5.3 Specimen-Specific Lysis & Challenging Matrices
6 Nucleic Acid Quantification & Electrophoresis
6.1 UV Spectrophotometry & Fluorometry 6.2 Agarose & Polyacrylamide Gel Electrophoresis 6.3 Capillary Electrophoresis & Microfluidic Bioanalyzers 6.4 Mass Spectrometry (MALDI-TOF) in Molecular Diagnostics
7 PCR Principles & Primer Design Optimization
7.1 Conventional PCR Thermodynamics & Cycling Kinetics 7.2 Primer Design Criteria, Melting Temp & Secondary Structures 7.3 PCR Inhibition, Facilitators & Troubleshooting
8 Advanced Amplification Technologies
8.1 Real-Time qPCR Probe Chemistries & Fluorescent Dyes 8.2 qPCR Quantification (Standard Curve, ΔΔCt) & Dissociation Melt Curve Analysis 8.3 Digital PCR (dPCR), Droplet Partitioning & Poisson Absolute Quantification 8.4 Isothermal & Target Multiplex Amplification
9 DNA Sequencing Technologies
9.1 Sanger Dideoxy Sequencing & Electropherogram Interpretation 9.2 NGS Library Preparation, Fragmentation & Target Enrichment 9.3 Massively Parallel NGS Platforms & Chemistries 9.4 NGS Quality Metrics, Coverage Depth & Bioinformatics
10 Hybridization, Blotting & Cytogenetic Assays
10.1 Southern, Northern & Dot Blot Chemistries 10.2 Fluorescence In Situ Hybridization (FISH) & CISH 10.3 Microarrays & Array Comparative Genomic Hybridization (aCGH)
11 Molecular Infectious Disease Diagnostics
11.1 Quantitative Viral Load Monitoring (HIV-1, HCV, HBV, CMV) 11.2 Qualitative Multiplex Syndromic Pathogen Panels 11.3 Antimicrobial Resistance Genotyping 11.4 Molecular Epidemiology & Pathogen Typing
12 Molecular Oncology & Hematopathology
12.1 Hematologic Malignancies & Translocation Assays 12.2 Solid Tumor Somatic Driver Mutations 12.3 Microsatellite Instability & DNA Mismatch Repair 12.4 Liquid Biopsy, ctDNA & Minimal Residual Disease
13 Medical Genetics, Pharmacogenomics & Identity Testing
13.1 Single-Gene Mendelian Disorders & CFTR Testing 13.2 Trinucleotide Repeat Expansion Disorders 13.3 Clinical Pharmacogenomics (PGx) Core Alleles 13.4 Human Identity Testing & Post-Transplant Chimerism
14 Quality Assurance, Controls & Method Validation
14.1 Quality Assurance Controls & Reference Materials 14.2 Clinical Method Validation & Performance Characteristics 14.3 Quality Management, Proficiency Testing & Competency
15 Laboratory Operations, Safety & Clinical Variant Reporting
15.1 Physical Facility Design & Unidirectional Workflow 15.2 Laboratory Biosafety, Chemical Hygiene & Waste 15.3 Clinical Calculations, ACMG Variant Classification & HGVS
8.4 Isothermal & Target Multiplex Amplification
Key Takeaways Explain This Topic AI
Isothermal nucleic acid amplification technologies synthesize millions of amplicon copies at a single constant operating temperature (37°C–65°C), eliminating thermal cycling hardware to enable ultra-rapid point-of-care (POC) and high-throughput clinical screening.
Transcription-Mediated Amplification (TMA) and NASBA operate at 41°C–42°C utilizing Reverse Transcriptase, RNase H, and T7 RNA Polymerase to produce 100–1,000 single-stranded RNA amplicons per template cycle, achieving ultra-high sensitivity for blood donor screening (HIV, HCV, HBV) and STIs.
Loop-Mediated Isothermal Amplification (LAMP) operates at 60°C–65°C using 4 to 6 specialized primers and strand-displacing Bst DNA Polymerase to generate multimeric cauliflower-like concatemers detectable via magnesium pyrophosphate turbidity or visual pH/colorimetric shifts.
Strand Displacement Amplification (SDA) utilizes restriction endonuclease hemiphosphorothioate nicking and exo- Bst polymerase, while Helicase-Dependent Amplification (HDA) uses DNA helicase (UvrD) to unwind duplexes enzymatically.
Clinical syndromic multiplex panels (BioFire FilmArray, Luminex xTAG, GenMark ePlex) amplify 15–30+ targets simultaneously, requiring universal-tailed primers, Tm harmonization, and nested microfluidics to prevent primer-dimer interference and reagent depletion.
Last updated: August 2026
8.4 Isothermal & Target Multiplex Amplification
Quick Summary: While conventional and real-time PCR rely on precise thermal cycling between denaturation ( 95 ∘ C 95^\circ\text{C} 9 5 ∘ C ), annealing ( 55 – 60 ∘ C 55–60^\circ\text{C} 55–6 0 ∘ C ), and extension ( 72 ∘ C 72^\circ\text{C} 7 2 ∘ C ), isothermal amplification technologies synthesize massive quantities of nucleic acid amplicons at a single, constant operating temperature ( 37 ∘ C– 65 ∘ C 37^\circ\text{C} – 65^\circ\text{C} 3 7 ∘ C –6 5 ∘ C ) . By replacing thermal denaturation with enzymatic mechanisms—such as T7 RNA transcription , strand-displacing polymerases ( Bst ) , restriction enzyme nicking , or DNA helicases —isothermal systems achieve rapid amplification ( 10 9 -fold in 15 – 45 minutes 10^9\text{-fold in } 15–45\text{ minutes} 1 0 9 -fold in 15–45 minutes ) on simplified, low-power instrumentation ideal for Point-of-Care (POC) diagnostics and ultra-high-throughput clinical screening ( Transcription-Mediated Amplification [TMA] , LAMP , SDA , HDA , RPA ). Simultaneously, multiplex target amplification enables syndromic testing for dozens of respiratory, gastrointestinal, and central nervous system pathogens in a single clinical sample using advanced nested microfluidic cartridge systems .
1. Isothermal Amplification Principles & Point-of-Care Utility
In standard PCR, thermal ramping between temperatures is often the rate-limiting step, requiring complex Peltier blocks, high electrical power, and heated lids. Isothermal amplification overcomes these constraints by operating continuously at a single temperature. +----------------------------------------------------------------------------------------------------+
- | ISOTHERMAL vs. THERMOCYCLED AMPLIFICATION |
- +-------------------+-----------------------------------+--------------------------------------------+
- | Parameter | Thermocycled PCR / qPCR | Isothermal Amplification (TMA, LAMP, SDA) |
- +-------------------+-----------------------------------+--------------------------------------------+
| **Thermal Profile**| Cyclical: $95^\circ\text{C} \rightarrow 55^\circ\text{C} \rightarrow 72^\circ\text{C}$ | **Constant single temperature** ($41^\circ–65^\circ\text{C}$) |
- +-------------------+-----------------------------------+--------------------------------------------+
- | **Strand** | Thermal heat ($95^\circ\text{C}$) breaks | **Enzymatic**: RNA degradation (RNase H), |
- | **Separation** | hydrogen bonds | strand displacement (*Bst*), nicking, or |
- | | | DNA helicase (*UvrD*) |
- +-------------------+-----------------------------------+--------------------------------------------+
- | **Amplification** | Binary doubling per cycle | **Continuous / Geometric transcription** |
- | **Kinetics** | ($2^n$ accumulation; ~1–2 hours) | ($10^9$-fold accumulation in 15–40 min) |
- +-------------------+-----------------------------------+--------------------------------------------+
- | **Amplicon Nature**| Double-stranded DNA (dsDNA) of | Single-stranded RNA (TMA/NASBA) or |
- | | fixed, discrete base-pair size | multimeric inverted concatemers (LAMP) |
- +-------------------+-----------------------------------+--------------------------------------------+
- | **Instrumentation**| Microprocessor thermal cycler with| Simple heating block, water bath, or |
- | | rapid Peltier heating/cooling | battery-powered POC handheld device |
- +-------------------+-----------------------------------+--------------------------------------------+
- 2. Transcription-Mediated Amplification (TMA) & NASBA
Transcription-Mediated Amplification (TMA) and Nucleic Acid Sequence-Based Amplification (NASBA) are transcription-based isothermal systems that amplify single-stranded RNA (ssRNA) targets at a constant temperature of 41 ∘ C– 42 ∘ C 41^\circ\text{C} – 42^\circ\text{C} 4 1 ∘ C –4 2 ∘ C . TRANSCRIPTION-MEDIATED AMPLIFICATION (TMA)
Step 1: Primer 1 (with T7 RNA Polymerase Promoter Tail) binds target ssRNA.
- 5'--[ T7 Promoter ]==================>
- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (Target (+) ssRNA)
- |
- v (M-MLV Reverse Transcriptase extends cDNA)
- 5'--[ T7 Promoter ]========================================== (cDNA strand)
- ------------------------------------------------------------ (Degraded by RNase H!)
- |
Step 2: Primer 2 binds cDNA; RT synthesizes second DNA strand.
- <==================== 5' (Primer 2)
- 5'--[ T7 Promoter ]==========================================
- |
- v
- 5'==[ ds T7 Promoter ]======================================= (Double-stranded DNA Template)
- |
Step 3: T7 RNA Polymerase binds promoter -> Generates 100 - 1,000 (-) ssRNA transcripts!
- [ T7 RNA Polymerase ] ===> ===> ===>
- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (100 - 1,000 ssRNA Amplicons
- ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ per DNA template!)
- The Enzymatic Cascade of TMA / NASBA
Enzyme Systems :
TMA (Hologic Aptima platforms) : Uses two enzymes— M-MLV Reverse Transcriptase (possessing both RNA-directed DNA polymerase activity and intrinsic RNase H activity) and T7 RNA Polymerase .
NASBA (bioMérieux NucliSENS platforms) : Uses three enzymes— AMV Reverse Transcriptase , exogenous E. coli RNase H , and T7 RNA Polymerase .
The Amplification Cycle :
Primer 1 Binding : A forward primer containing a 5' bacteriophage T7 promoter sequence binds to the target RNA.
cDNA Synthesis & Template Degradation : Reverse Transcriptase synthesizes a complementary DNA (cDNA) strand. Intrinsic RNase H selectively degrades the original RNA template from the RNA:DNA heteroduplex.
Double-Stranded Promoter Formation : Primer 2 binds the single-stranded cDNA, and Reverse Transcriptase extends it to create a fully double-stranded DNA template containing an active, functional double-stranded T7 promoter.
High-Yield Autocatalytic Transcription : T7 RNA polymerase binds the promoter and transcribes 100 to 1 , 000 single-stranded RNA amplicons 100\text{ to } 1,000\text{ single-stranded RNA amplicons} 100 to 1 , 000 single-stranded RNA amplicons from each DNA template. Each newly synthesized RNA amplicon immediately enters the cycle as a template for Primer 2, driving exponential amplification that yields > 10 9 >10^9 > 1 0 9 -fold amplification in under 45 minutes.
Clinical Diagnostic Applications of TMA
Blood Donor Screening : Ultra-sensitive screening of pooled donor blood for HIV-1 RNA, HCV RNA, HBV DNA, West Nile Virus (WNV), and Zika Virus .
Sexually Transmitted Infections (STIs) : Gold standard testing on the Hologic Panther system for Chlamydia trachomatis , Neisseria gonorrhoeae , Trichomonas vaginalis , and Mycoplasma genitalium .
Oncology : Detection of oncogenic HPV E6/E7 mRNA transcripts, which correlate with active cervical dysplasia more specifically than HPV DNA testing.
3. Loop-Mediated Isothermal Amplification (LAMP)
Loop-Mediated Isothermal Amplification (LAMP) is an ultra-high-efficiency isothermal technology operating at 60 ∘ C– 65 ∘ C 60^\circ\text{C} – 65^\circ\text{C} 6 0 ∘ C –6 5 ∘ C that produces up to 10 10 10^{10} 1 0 10 amplicon copies within 20 – 30 minutes 20–30\text{ minutes} 20–30 minutes . LAMP PRIMER DESIGN & TARGET REGIONS
- [ F3c ] [ F2c ] [ F1c ] [ B1 ] [ B2 ] [ B3 ]
- 5' --------+----------+----------+----------------+---------+---------+-------- 3'
- 3' --------+----------+----------+----------------+---------+---------+-------- 5'
- [ F3 ] [ F2 ] [ F1 ] [ B1c] [ B2c] [ B3c]
Primers:
- FIP (Forward Inner Primer): [ F1c sequence ] + [ F2 sequence ]
- BIP (Backward Inner Primer): [ B1c sequence ] + [ B2 sequence ]
- F3 (Forward Outer Primer): Displaces FIP-extended strand
- B3 (Backward Outer Primer): Displaces BIP-extended strand
- Loop Primers (LoopF / LoopB): Accelerate amplification by priming stem loops
- The Mechanism of LAMP
Enzyme : Utilizes Bst DNA Polymerase (Large Fragment) purified from Bacillus stearothermophilus , which possesses powerful 5 ′ → 3 ′ 5' \rightarrow 3' 5 ′ → 3 ′ strand-displacement activity and lacks 5 ′ → 3 ′ 5' \rightarrow 3' 5 ′ → 3 ′ exonuclease activity.
Primer Design Architecture : Employs 4 to 6 specialized primers recognizing 6 to 8 distinct regions on the target sequence:
Forward Inner Primer (FIP) : Hybridizes via its F2 region. Its 5' F1c tail is complementary to the downstream F1 region.
Outer Primers (F3 and B3) : Anneal upstream of the inner primers; extension by Bst displaces the newly synthesized FIP/BIP strands.
Dumbbell Stem-Loop Formation : The displaced single-stranded DNA folds back on itself as F1c pairs with F1 (and B1c with B1), creating a self-annealing dumbbell-shaped structure .
Concatemer Synthesis : The 3' end of the dumbbell serves as a self-primer for continuous elongation, generating large, repeating multimeric cauliflower-like concatemers of alternating inverted repeats.
Loop Primers (LoopF and LoopB) : Bind to loops not involved in self-priming, speeding up the reaction from 60 minutes down to 15 – 20 minutes 15–20\text{ minutes} 15–20 minutes . DUMBBELL STRUCTURE IN LAMP
- /---\ /---\
- / \ / \
- | F1c |===========| B1 |
- \ / \ /
- \---/ (F1) (B1c)\---/
- Detection Chemistries for LAMP
Magnesium Pyrophosphate Turbidity : As Bst incorporates hundreds of thousands of dNTPs, massive quantities of inorganic pyrophosphate ( P 2 O 7 4 − \text{P}_2\text{O}_7^{4-} P 2 O 7 4 − ) are released, reacting with Mg 2 + \text{Mg}^{2+} Mg 2 + in the buffer to precipitate insoluble magnesium pyrophosphate ( Mg 2 P 2 O 7 \text{Mg}_2\text{P}_2\text{O}_7 Mg 2 P 2 O 7 ) , producing visible white turbidity measured in real time.
Metal-Chelating Dyes (Calcein / Hydroxynaphthol Blue [HNB]) : Calcein fluorescence is initially quenched by binding manganese ( Mn 2 + \text{Mn}^{2+} Mn 2 + ). As pyrophosphate is generated, it strips Mn 2 + \text{Mn}^{2+} Mn 2 + from calcein, allowing it to bind Mg 2 + \text{Mg}^{2+} Mg 2 + and emit bright visual green fluorescence.
pH-Sensitive Colorimetric Shifts (Phenol Red) : DNA polymerase extension releases one proton ( H + \text{H}^+ H + ) per incorporated nucleotide ( dNTP → dNMP + PPi + H + \text{dNTP} \rightarrow \text{dNMP} + \text{PPi} + \text{H}^+ dNTP → dNMP + PPi + H + ). In a weakly buffered master mix, the release of millions of protons drops the reaction pH from 8.8 8.8 8.8 to < 6.5 <6.5 < 6.5 , inducing an instantaneous visual color change in phenol red from pink/red (negative) to bright yellow (positive) .
4. Other Isothermal Technologies: SDA, HDA & RPA +----------------------------------------------------------------------------------------------------+
- | SDA, HDA & RPA COMPARISON |
- +-------------------+--------------------+--------------------+--------------------------------------+
- | Isothermal Assay | Operating Temp | Primary Enzymes | Molecular Mechanism & Clinical Assay |
- +-------------------+--------------------+--------------------+--------------------------------------+
- | **Strand** | $37^\circ–52^\circ\text{C}$| • Restriction | Primers incorporate a hemiphosphoro- |
- | **Displacement** | (Thermophilic SDA | Endonuclease | thioate restriction site (*HincII*). |
| **Amplification** | at $52^\circ–55^\circ\text{C}$) | (*HincII* or *BsoBI*)| Enzyme nicks un-modified strand; |
- | **(SDA)** | | • *exo- Bst* DNA | *exo- Bst* extends from nick, |
- | | | Polymerase | displacing downstream strand. |
- | | | • $\text{dATP}\alpha\text{S}$ modified| **Clinical: BD Viper (CT/NG)** |
- +-------------------+--------------------+--------------------+--------------------------------------+
- | **Helicase-** | $60^\circ–65^\circ\text{C}$| • Thermostable DNA | A DNA helicase (*UvrD*) unwinds the |
- | **Dependent** | (Thermophilic tHDA)| Helicase (*UvrD*) | DNA duplex enzymatically, eliminating|
- | **Amplification** | | • Strand-displacing| thermal denaturation. Primers bind |
- | **(HDA)** | | DNA Polymerase | ssDNA; polymerase extends. |
- | | | • Accessory cofactors| **Clinical: Quidel Solana / AmpliVue**|
- +-------------------+--------------------+--------------------+--------------------------------------+
- | **Recombinase** | **$37^\circ–42^\circ\text{C}$**| • T4 Recombinase | Recombinase (UvsX) + UvsY loads onto |
- | **Polymerase** | (Body temperature; | (UvsX / UvsY) | primers, scanning dsDNA to invade |
- | **Amplification** | rapid 15-min POC) | • Single-Stranded | homologous duplexes. *Bsu* polymerase|
- | **(RPA)** | | Binding (gp32) | extends; SSB stabilizes D-loop. |
- | | | • *Bsu* DNA Pol | **Clinical: Lateral flow POC strips**|
- +-------------------+--------------------+--------------------+--------------------------------------+
- 5. Target Multiplex Amplification & Syndromic Panel Design
Clinical diagnostic microbiology has transitioned toward syndromic multiplex testing , simultaneously interrogating 15 to 30+ potential pathogens from a single clinical specimen (e.g., Upper Respiratory Panels, Gastrointestinal Panels, Meningitis/Encephalitis Panels). SYNDROMIC MULTIPLEX PANEL ARCHITECTURE
- Clinical Specimen (e.g., Nasopharyngeal Swab in VTM)
- |
- v
- [ High-Plex Microfluidic Cartridge (e.g., BioFire FilmArray) ]
- +----------------------------------------------------------------------+
- | Stage 1: Low-Cycle Multiplex Pre-Amplification (Outer Primers) |
- | Enriches all 20+ viral/bacterial targets simultaneously |
- +----------------------------------+-----------------------------------+
- |
- v (Aliquoted into micro-wells)
- +----------------------------------+-----------------------------------+
- | Stage 2: Single-Plex High-Resolution Inner Nested PCR |
- | Micro-well 1: Influenza A Micro-well 5: SARS-CoV-2 |
- | Micro-well 2: Influenza B Micro-well 6: RSV A/B |
- | Micro-well 3: Adenovirus Micro-well 7: Human Metapneum|
- | Micro-well 4: Rhinovirus Micro-well 8: Bordetella pert|
- +----------------------------------------------------------------------+
- The Mathematics & Challenges of Multiplex Primer Design
Primer Interaction Formula : In a multiplex assay containing N N N individual primer pairs ( 2 N 2N 2 N total primer oligonucleotides), the number of potential pairwise primer-primer interactions ( I I I ) scales quadratically: I = 2 N ( 2 N − 1 ) 2 = N ( 2 N − 1 ) I = \frac{2N(2N - 1)}{2} = N(2N - 1) I = 2 2 N ( 2 N − 1 ) = N ( 2 N − 1 ) For a 20-target panel (40 primers), there are 40 ( 39 ) / 2 = 780 potential primer-primer cross-hybridizations 40(39)/2 = 780\text{ potential primer-primer cross-hybridizations} 40 ( 39 ) /2 = 780 potential primer-primer cross-hybridizations that can form non-specific primer-dimers!
Amplicon Competition & Bias : Amplicons with lower GC content, shorter lengths, or higher initial template concentrations amplify faster, rapidly exhausting master mix dNTPs and Mg 2 + \text{Mg}^{2+} Mg 2 + cations while starving longer or GC-rich targets.
Multiplex Optimization Strategies
T m T_m T m Harmonization : All primer melting temperatures must be strictly matched within ± 1.0 ∘ C \pm 1.0^\circ\text{C} ± 1. 0 ∘ C (typically 58 ∘ – 60 ∘ C 58^\circ–60^\circ\text{C} 5 8 ∘ –6 0 ∘ C ).
Universal-Tailed Primers : Primers are synthesized with a target-specific 3' sequence and a common universal 5' tail sequence . After the first two cycles, all amplicons incorporate the universal tail, allowing subsequent cycles to be driven by a single universal primer pair, equalizing amplification efficiencies across all targets.
Two-Stage Nested Microfluidics (BioFire FilmArray) :
Stage 1 (Multiplex Outer PCR) : A low-cycle (15–18 cycles) bulk multiplex PCR enriches all targets using outer primer pairs.
Stage 2 (Nested Inner Single-Plex PCR) : The diluted Stage 1 product is pumped across an array of hundreds of individual micro-wells, each containing a single inner nested primer pair for real-time fluorescence and melt curve identification.
6. Comprehensive Comparative Synthesis of Isothermal Platforms
Platform / Assay Operating Temp Enzymes Employed Primers Required Amplicon Type Primary Clinical Diagnostic Tests
Transcription-Mediated Amplification (TMA) 41 ∘ – 42 ∘ C 41^\circ–42^\circ\text{C} 4 1 ∘ –4 2 ∘ C M-MLV RT (RNase H) + T7 RNA Polymerase 2 (Primer 1 with T7 promoter tail) Single-stranded RNA ( 100 – 1 , 000 copies/cycle 100–1,000\text{ copies/cycle} 100–1 , 000 copies/cycle ) Blood screening (HIV/HCV/HBV/WNV), C. trachomatis , N. gonorrhoeae , HPV mRNA
Nucleic Acid Sequence-Based Amplification (NASBA) 41 ∘ C 41^\circ\text{C} 4 1 ∘ C AMV RT + E. coli RNase H + T7 RNA Polymerase 2 (T7 promoter primer) Single-stranded RNA Enterovirus, CMV pp67 mRNA, respiratory viral loads
Loop-Mediated Isothermal Amplification (LAMP) 60 ∘ – 65 ∘ C 60^\circ–65^\circ\text{C} 6 0 ∘ –6 5 ∘ C Strand-displacing Bst DNA Polymerase 4 to 6 (FIP, BIP, F3, B3, LoopF, LoopB) Multimeric concatemers with stem loops Rapid POC SARS-CoV-2, malaria, tuberculosis, norovirus
Strand Displacement Amplification (SDA) 37 ∘ – 52 ∘ C 37^\circ–52^\circ\text{C} 3 7 ∘ –5 2 ∘ C Restriction Endonuclease ( HincII ) + exo- Bst Pol 4 (2 bumper + 2 nicking primers) Single-stranded DNA fragments BD Viper C. trachomatis / N. gonorrhoeae
Helicase-Dependent Amplification (HDA) 60 ∘ – 65 ∘ C 60^\circ–65^\circ\text{C} 6 0 ∘ –6 5 ∘ C DNA Helicase ( UvrD ) + Strand-displacing DNA Pol 2 standard primers Double-stranded DNA amplicons Quidel Solana / AmpliVue C. difficile , Group A Strep, HSV 1/2
Recombinase Polymerase Amplification (RPA) 37 ∘ – 42 ∘ C 37^\circ–42^\circ\text{C} 3 7 ∘ –4 2 ∘ C T4 UvsX / UvsY + gp32 SSB + Bsu DNA Polymerase 2 standard primers Double-stranded DNA amplicons Handheld field diagnostics, Ebola, Dengue, biothreat pathogen detection