Isothermal Amplification

IsoPol™ BST+ is well suited for isothermal applications such as LAMP and RT-LAMP for its superior amplification performance and robustness.

Isothermal amplification techniques are often a better choice than polymerase chain reaction for point-of-care (POC) diagnostics in areas with limited laboratory infrastructure resources and trained personnel.

Some of the most frequently used techniques are loop-mediated amplification (LAMP), strand-displacement amplification (SDA) and helicase-dependent amplification (HDA).

Applying these techniques has resulted in a more rapid, sensitive, accurate, simpler and cost-effective procedure for detection of nucleic acid from samples.

Learn more about IsoPol™ BST⁺

LAMP / RT-LAMP

LAMP is widely used in laboratories for rapid detection of pathogenic agents (bacteria, parasites, and viruses) causing disease. Because of its simplicity, sensitivity and speed, the method is key for point-of-care diagnostics settings.

LAMP replicates nucleic acids at a single temperature by utilizing a DNA polymerase with high strand displacement, e.g. Bacillus stearothermophilus (Bst) DNA polymerase.

For amplification of RNA based targets, a one-step reverse transcriptase is added to the LAMP reaction to synthesize complementary DNA from RNA sequences without performing additional DNA purification step, i.e. reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

Strand Displacement Activity

IsoPol™ BST+ is engineered for stronger strand displacement activity, increasing the amplification speed and robustness.

LAMP amplification was performed using primer sequences detecting DNA from the Lambda virus.

All polymerases were tested using the corresponding 10X isothermal amplification buffer according to manufacturer’s recommendations.

Reaction mixtures (30 µl total volume) contained: 0.32 U/µl of DNA polymerase (BST+, an engineered Bst from Vendor A, or Bst Large Fragment (LF)), 6 mM MgSO₄, 1.4 mM dNTP (each), 1.3 μM EvaGreen® Fluorescent DNA Stain, primers: 800 nM FIP+BIP, 200 nM F3+B3, 400 nM LF+LB, and Lambda DNA template (0.05, 0.5 or 5 ng).

LAMP reactions were performed at 65°C for 30 min and analysed on Genie® II platform.

The results as illustrated in figure 1, demonstrate that IsoPol BST+ provides superior time to results compared to a market leading polymerase used in LAMP assay development.

Figure 1. IsoPol BST+ provides faster amplification performance compared to both the native Bst polymerase and an engineered version from another supplier. All NTCs remained negative.

RT-LAMP amplification was performed by combining an Avian Myeloblastosis Virus Reverse Transcriptase (AMV-RT) with the relevant polymerase using RNA from bacteriophage MS2 as template.

All polymerases were tested using the corresponding 10X isothermal amplification buffer according to manufacturer’s recommendations.

Reaction mixtures (30 µl total volume) contained: 0.32 U/µl of DNA polymerase (BST+, an engineered Bst from Vendor A, or Bst Large Fragment (LF)), 8 mM MgSO4, 1.4 mM dNTP (each), 1.3 μM EvaGreen® Fluorescent DNA Stain, primers: 800 nM FIP+BIP, 200 nM F3+B3, 400 nM LF+LB, 0.105 U/µl AMV-RT and 10 ng MS2 RNA template.

RT-LAMP reactions were performed at 65°C for 45 min and analysed on Genie® II platform.

Figure 2 demonstrates that IsoPol BST+ provides superior time-to-results in RT-LAMP.

Figure 2. IsoPol BST+ provides faster amplification performance compared to standard Bst polymerases when using 10 ng MS2 RNA template and AMV-RT in RT-LAMP. All NTCs were negative. n=2.

Serum Tolerance

IsoPol™ BST+ robustness, superior speed and high salt tolerance makes it the ideal companion polymerase of choice for POC-LAMP/RT-LAMP testing.

IsoPol™ BST+ has a higher salt tolerance than most Bst-derived polymerases, which indicates high tolerance to common matrix inhibitors.

LAMP amplification was performed using DNA detecting the Lambda DNA virus.

All polymerases were tested using the corresponding 10X isothermal amplification buffer according to manufacturer’s recommendations.

Various amounts of human serum were added to the reaction to evaluate how each polymerase was affected by the presence of a biological matrix.

Reaction mixtures (30 µl total volume) contained: 0.32 U/µl of DNA polymerase (BST+, an engineered Bst from Vendor A, or Bst Large Fragment (LF)), 6 mM MgSO4, 1.4 mM dNTP (each), 1.3 μM EvaGreen® Fluorescent DNA Stain, primers: 800 nM FIP+BIP, 200 nM F3+B3, 400 nM LF+LB, human serum (0, 2, 5 or 10%), and 1 ng Lambda DNA template.

LAMP reactions were performed at 65°C for 40 min (1st run)/60 min (2nd run) and analysed on Genie® II platform.

Figure 3 demonstrates that IsoPol™ BST+ is an excellent choice for the detection of pathogens using LAMP directly in presence of biological matrices as human serum. A market leading, engineered Bst polymerase is heavily inhibited even in presence of small amounts of human serum.

 

Figure 3. IsoPol BST+ provides superior amplification performance in LAMP compared to other commercially available engineered Bst polymerases in the presence of serum.

 

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