AcuGenix™ Hot Start RT-LAMP Dye Master Mix-V2

Dye-based hot start RT-LAMP master mix with Neoscript Reverse Transcriptase for qualitative RNA detection at 60–65 °C in 30–60 minutes.

Lyo-ready FHW1008-R02

Lyo-ready

Beads and in-situ lyophilized powder

Bst 2.0 HS

Reversible modification, room-temp setup

15 freeze-thaw cycles

Stable amplification

AcuGenix™ Hot Start RT-LAMP Dye Master Mix-V2

AcuGenix™ Lyo-Ready Hot Start RT-LAMP Dye Master Mix-V2

Product overview

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technology. This kit adopts the dye-based isothermal amplification method and can be used for the qualitative detection of RNA in samples (e.g., viruses).

Available versions

Cat. no.	Product name	Formulation
HW206-R02	AcuGenix™ Hot Start RT-LAMP Dye Master Mix-V2	Liquid (with glycerol)
FHW1008-R02	AcuGenix™ Lyo-ready Hot Start RT-LAMP Dye Master Mix-V2	Lyo-ready

Features and applications

Features

Suitable for RNA sample detection by LAMP using intercalating dyes, requiring fluorescence detection instruments.
Reactions can be assembled at room temperature, effectively preventing non-specific amplification and enhancing reaction efficiency.
Specially developed for lyophilization processes, compatible with fully-formulated lyophilized product preparation such as lyophilized beads and in-situ lyophilized powder.
Engineered with reversible modification technology based on Bst 2.0. Enzyme activity is completely blocked at room temperature, allowing reaction setup under ambient conditions to suppress non-specific amplification and improve reaction efficiency. No separate activation step is required.
Adopts optimized LAMP dye method buffer dedicated for lyophilization.
Supports dye-based LAMP for qualitative detection.

Applications

Qualitative detection of DNA/RNA from samples including viruses.
Preparation of all-in-one lyophilized LAMP products (lyophilized beads, in-situ lyophilized powder).

Protocol

The protocol below is for the Liquid (with glycerol) format, HW206-R02. The Lyo-ready format, FHW1008-R02, uses a different reaction mix; refer to the FHW1008-R02 instruction manual in Downloads.

Prepare Reaction Mix

Components	25 µL System	50 µL System	Final Concentration
8 U/µL AcuGenix™ Bst Hot Start DNA Polymerase-V2	1 µL	2 µL	0.32 U/µL
200 U/µL Neoscript Reverse Transcriptase	0.375 µL	0.75 µL	3 U/µL
2×RT-LAMP Premix Buffer Ⅱ	12.5 µL	25 µL	1×
50×RT-LAMP Fluorescent Dye Ⅱ	0.5 µL	1 µL	1×
10×Primer Mix¹	2.5 µL	5 µL	1×
Template RNA²	——	——	——
ddH₂O	To 25 µL	To 50 µL	——

Recommended primer concentration: The 10× Primer Mix comprises 16 µM FIP, 16 µM BIP, 2 µM F3, 2 µM B3, 4 µM LoopF, and 4 µM LoopB. Concentration gradient testing can be carried out to determine the optimal primer dosage. It is recommended that primers be prepared with TE Buffer.
Different types of RNA templates have different copy numbers of target genes. When necessary, gradient dilution can be performed to determine the optimal quantity of RNA template to be added.

Reaction Conditions

Incubate at a temperature range of 60–65 °C for a duration of 30–60 minutes, and acquire fluorescence data at one-minute intervals. After the reaction is completed, allow the sample to cool to ambient temperature before analyzing the results.

Q&A

LAMP amplification products are scarce or non-existent:

Elevate the quantity of the template and the concentration of the enzyme.
Substitute the primers. It is advisable to design multiple primer sets and conduct screening for the optimal primers via preliminary experiments. For primer design, refer to http://primerexplorer.jp/e/.
The use of Loop Primers is recommended to enhance amplification efficiency and reduce amplification time.

Components

Component	Specification/Notes
Liquid (with glycerol), HW206-R02
8 U/µL AcuGenix™ Bst Hot Start DNA Polymerase-V2	N/A
200 U/µL Neoscript Reverse Transcriptase	Included component; refer to the instruction manual
2×RT-LAMP Premix Buffer Ⅱ	Included component; refer to the instruction manual
50×RT-LAMP Fluorescent Dye Ⅱ	Included component; refer to the instruction manual
Lyo-ready, FHW1008-R02
5×RT-LAMP Lyophilization Buffer	5×
10×RT-LAMP Premix (DG) (Bst2.0 HS)	10×
50×RT-LAMP Fluorescent Dye Ⅱ	50×

Performance data

1. Freeze-Thaw Stability Test

Channel	1ng/T	0.1ng/T	0.01ng/T	NTC
Control 5 freeze-thaw cycles 10 freeze-thaw cycles 15 freeze-thaw cycles
FAM

NTC Testing Standard:NTC amplification start time >30 min;

Conclusion 1: FHW1008-R02 were subjected to 5, 10, or 15 freeze-thaw cycles and compared with the test group stored at -20°C. It demonstrates stable amplification after 5, 10, or 15 freeze-thaw cycles.

2. Comparison with Competitive Reagents

channel	10 copies/T	5 copies/T
FHW1008-R02 Supplier A
FAM

Conclusion 1: Compare the amplification performance differences between FHW1008-R02 and Supplier A in the pet pathogen detection model.The detection sensitivity and uniformity of FHW1008-R02 are superior to Supplier A.