Attogene NA2024 Microcystin qPCR Detection Kit Instructions

: June 13, 2024
: Attogene

Table of Contents

Background
Test Principle
Applications
Equipment and Reagents Needed (not provided)
Components Provided in This Kit
Reagents Preparation
Control Preparation
Assay Set Up
qPCR detection protocol
qPCR Amplification Protocol
Expected Performance
Interpreting the test results
General Instructions
Storage
References
Read User Manual Online (PDF format)
Download This Manual (PDF format)

Microcystin qPCR detection Kit
Real-time quantitative analysis of the Microcystis toxin gene region MycE
Catalog Number: NA2024

For Research Use Only. Not for use in Diagnostic Procedures.

Background

Microcystis are a class of hepatotoxins produced by blue-green algae such as Microcystis aeruginosa. Microcystin-LR is the most common of the over 50 different congeners. Cyanobacteria can produce microcystin in large quantities during an algal bloom, which then poses a major threat to our aquatic ecosystems and sources of food, as there is a well-documented phenomenon called “bioaccumulation” that describes the increase in consumed toxin quantities as trophic levels increase.

Test Principle

Attogene’s PCR kit for Microcystin is designed for the In vitro analysis of the MycE gene region responsible for assembling part of the Microcystis peptide. The MycE gene region specific primer and probe mix is provided to be detected through the FAM channel on a qPCR machine. A sample of algae is obtained and washed to extract a clean algal gDNA sample. A reaction mixture is assembled from primers, probe, master mix, and gDNA samples as required. The qPCR machine of choice is set up and loaded as needed and the mixture undergoes PCR amplification. The Primer mix provided exploits the Taq polymerase to amplify the gene region of interest; while the DNA probe mixture is cleaved during amplification to release its FAM fluorophore. The resulting FAM release can be detected on a variety of qPCR platforms.

Applications

This kit can be used for specific analysis of the Microcystis MycE gene region in liquid gDNA samples such as water, wastewater, and algal cultures.

Equipment and Reagents Needed (not provided)

Real-time PCR Instrument
qPCR 2X Master Mix
DNA extraction kit/ gDNA sample
PCR reaction tubes/plate
Vortex and centrifuge
Polystyrene centrifuge tube: 50mL, 2mL
Micropipettes & Tips

Components Provided in This Kit

MycE region specific primer mixture
MycE region specific fluorescent probe
MycE region specific positive control template (Standard curve)
RNase/DNase free water (resuspension of positive control template and standard curve preparation)

Reagents Preparation

Master Mix (not Provided)

Store at -20C. Master Mix is stable when stored at -20C. Freeze and thaw cycles should be minimized to 6 cycles to increase the shelf life.
If required aliquots of Master Mix should be made and stored at -20C to minimize freeze thaw.

Primer/ Probe Mixture

Precautions: these reagents are light sensitive and should only be handled in a clean area
Store at -20C. Primer/Probe is stable when stored at -20C. Freeze and thaw cycles should be minimized to 6 cycles to increase the shelf life.
If required aliquots of Primer/Probe should be made and stored at -20C in the dark to minimize freeze thaw.

Control Preparation

Negative extraction control (NEC)

Prepare at least one NEC each time extracting DNA from your sample.
RNase/DNase free water is used in place of algae in the extraction system to create a negative for the DNA isolation method.
The NEC will serve as a contamination control method for the isolation.

No Template control (NTC)

If necessary, a NTC can made by replacing gDNA in the PCR reaction with RNase/DNase free water
The NTC is used to check for contamination during PCR plate set up

Positive control template (PCT)

The MycE Positive control template contains a high number of copy templates and should be handled away from specimen samples and kit components.
The PCT contains synthetic Microcystin DNA representing the MycE genomic region of interest.
To ensure PCR run validity, the PCT should see amplification in the FAM channel.
Store at -20C. PCT is stable when stored at -20C. Freeze and thaw cycles should be minimized to 6 cycles to increase the shelf life.

Assay Set Up

gDNA isolation will need to be done before starting an experiment. For optimal results use >10ng/uL of gDNA with a ratio of >1.80 in your experiment. IEC multiplexing can also be done to ensure proper DNA extraction.
Plate set up will vary with the quantity of samples you need to run on your plate. A NEC is preferably included in each plate set up. NTCs should be included in each plate set up. A PCT must be included in each plate set up. The PCT should be added after all other reagents and samples have been added to the plate.
Determine the number of reactions to set up in your assay (including NEC, PCT and any NTCs for your plate). It is necessary to make extra reaction mixture to allow for pipetting error.
For convenience a large solution of PCR components will be mixed shortly before starting a reaction and subsequently aliquoted into your plate or tubes. Each PCR run will use 18uL of this reaction mixture and 1ul of isolated gDNA/NEC/PCT/NTC based on the experiment set up.

qPCR detection protocol

For each DNA sample prepare a reaction mix according to the table below:
(Include sufficient reactions for positive and negative controls)

Reagent	Quantity
2X Master Mix	10uL
PCR Water	6uL
Mc Probe	1uL
Dual Mc Primer set	1uL
Final Volume	18uL

Pipette 15uL of this mixture into each well according to your qPCR experimental plate set up.
Prepare your DNA templates for each reaction
Pipette 2uL of sample gDNA into each well, according to your experiment. For negative controls replace the gDNA sample with 2uL of RNase/DNase free water to bring the total volume to 20uL.
To prepare a standard curve dilution series.

Pipette 40uL of RNase/DNase free water into 5 tubes and label 2-6
Pipette 10uL of Positive Control Template into tube 2
Vortex thoroughly
Pipette 10ul from tube 2 into tube 3
Vortex thoroughly

Repeat steps 4-5 Standard Curve	Copy Number
Tube 1 (Positive Control Template)	2×10^5 per uL
Tube 2	4×10^4 per uL
Tube 3	8×10^3 per uL
Tube 4	1600 per uL
Tube 5	320 per uL
Tube 6	64 per uL

Use 2ul of standard curve template into each well according to your experiment and plate set up.
Final well volume should be 20uL.

qPCR Amplification Protocol

Amplification conditions using 2x qPCR Master Mix:

Steps	Time	Temperature	Cycles	Detection Format

Initial Denaturation
(Taq Activation)| 2 Minutes| 94C| 1| MycE = FAM (456-510)
Denaturation| 20 sec.| 94C| 40
Annealing*| 30 sec.| 50C
Extension| 60 sec.| 14C

*Fluorogenic data should be read during this step through the FAM channel

Expected Performance

Before Interpreting results, it is necessary to verify the integrity of the reaction. If the following criteria are not satisfied, then testing needs to be repeated.
a. NEC is free from amplification in the FAM (465-510) channel.
b. NTC is free from amplification in the FAM (465-510) channel.
c. PCT produces amplification in the FAM (465-510) channel between CT 16-25
Manually inspect amplification criteria are fulfilled for all samples to verify the integrity of the results.

Interpreting the test results

If all the data analysis criteria are fulfilled, then each sample can be assessed with the following metric:

Target	Positive controt	Negative control	Result
+	+	–	Positive Quantitative result (calculate quantity)
–	+	–	Negative result
+/-	+	CT<35	Experiment failed (contamination)
+1-	+	CT>35	*
+/-	–	+/-	Experiment failed