Nucleic Acid Sequence–Based Amplification (NASBA) Prospective.pptx

Nucleic Acid Sequence–Based Amplification (NASBA) Prospective

1. Nucleic Acid Sequence–Based Amplification ( NASBA) Prospective and Applications By: Lamees Zaro

2. What is NASBA? NASBA: Nucleic Acid Sequence-Based Amplification . Developed by Compton (1991) as an alternative to PCR for RNA amplification . Uses three key enzymes : Reverse Transcriptase (AMV-RT): Converts RNA to cDNA. RNase H: Degrades RNA strand in RNA-DNA hybrids. T7 RNA Polymerase: Amplifies RNA exponentially from cDNA . Works at a constant temperature (41°C) for RNA amplification. 2

3. Characteristics of NASBA Isothermal amplification at 41°C (No thermocycler required). Specially suited for RNA targets like mRNA and rRNA . Produces single-stranded RNA, enabling direct detection . High sensitivity and specificity . Eliminates need for expensive thermocycling equipment. 3

4. Principles of NASBA P1 with a T7 RNA polymerase promoter is hybridized to the RNA target . Reverse transcriptase converts RNA to cDNA RNase H degrades RNA in the RNA-DNA hybrid. P2 binds cDNA and is extended by reverse transcriptase, forming double-stranded DNA with a T7 promoter . T7 RNA polymerase recognizes the promoter sequence and synthesizes multiple RNA copies from the double-stranded DNA template. Process repeats, exponentially amplifying RNA . 4

5. Primer Design Forward primer must have a T7 promoter sequence at the 5' end . Primers should hybridize to conserved RNA regions. Hybridizing region of primers should be 20–30 bases with 40–60% G/C content. 5

6. Optimization NASBA assay must be optimized prior to application. KCl Concentration : Critical for primer specificity, sensitivity, and yield . Optimal concentration: 70 mM (range: 50–90 mM ). Low concentrations: Increase false positives and primer-dimer formation . High concentrations: Inhibit primer annealing . Incubation time (90 minutes typical ). Primer concentration (~0.2 µM commonly used). 6

7. Detection Methods Traditional method: Agarose gel electrophoresis with ethidium bromide. Modern approaches : Enzyme-linked gel assay (ELGA): Horseradish peroxidase probe labeled Fluorescence correlation spectroscopy: fluorescent labeled probe, the increase in diffusion time is used to quantify the amplicon. Molecular beacons: Real-time fluorescence based detection during amplification. 7

8. Advantages of NASBA Amplifies over 10⁹ copies of nucleic acids in 90 minutes. Isothermal reaction at 41°C , no thermal cycler needed . Produces single-stranded RNA, ready for detection or re-amplification. Selectively amplifies RNA even with DNA background . Faster and more efficient with exponential RNA amplification . Avoids additional reverse transcription (RT) step, reducing contamination and hands-on time. Ideal for detecting RNA viruses, retroviruses, and gene expression . 8

9. Disadvantages of NASBA RNA degradation is a major limitation, requiring careful sample handling . Requires a melting step before amplification . Reaction temperature cannot exceed 42°C due to enzyme sensitivity . Effective for RNA targets only between 120–250 nucleotides . 9

10. Comparison with PCR NASBA vs. PCR : Isothermal vs. thermocycling. RNA specificity vs. DNA-focused. Faster amplification (90 minutes for NASBA). 10

11. R apid detection of viable  E.coli  in water using real-time NASBA The NASBA assay targets the clpB mRNA, which encodes a heat-shock protein specific to E. coli . mRNA is an excellent marker for viability since it degrades quickly in dead cells under environmental conditions. NASBA selectively amplifies RNA without interference from DNA, making it ideal for detecting live cells. 11

12. Rapid detection of viable  E.coli  in water using real-time NASBA Comparable to culture methods, capable of detecting as few as 1 viable E. coli per 100 mL of water when a heat-shock is applied . Heat-shock enhances the expression of clpB mRNA in viable cells, increasing detection efficiency. Does not produce false positives from dead cells or contamination, which can occur with PCR. 12

13. Comparison to Other Methods Traditional Culture : Reliable but slow, taking 18–24 hours to provide results . PCR: Faster but detects DNA from both viable and non-viable cells, potentially leading to false positives . NASBA: Combines the advantages of speed, sensitivity, and the ability to detect only viable cells. 13

14. Applications in Infectious Diseases Campylobacter spp .: NASBA targets 16S rRNA, providing faster and more reliable pathogen detection. 14

15. Respiratory Pathogen Detection Mycoplasma pneumoniae : Comparable sensitivity and specify to PCR . Chlamydia pneumoniae & Legionella pneumophila : Detects RNA using 16S rRNA . M. tuberculosis : Detects 16S rRNA from as few as 200 bacteria, demonstrate high sensitivity. Sensitive for viruses like Respiratory syncytial virus , parainfluenza , and influenza . 15

16. Challenges Struggles to detect mRNA from certain bacteria (e.g. Mycobacterium avium ssp. Paratuberculosis (MAP)). NASBA is not yet widely used in food analysis. Isothermal process, but detection requires complex equipment . More complex than PCR, limiting routine use in food/environmental diagnostics . 16

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