LyoDt® Real-Time PCR Detection Reagent for Cronobacter Freeze-Dried powder

LyoDt® Real-Time PCR Detection Reagent for Cronobacter Freeze-Dried powder

1. INTRODUCTION

Cronobacter is a newly defined genus of bacteria, whose predecessor is Enterobacter sakazakii. Cronobacter is a severe foodborne pathogen primarily transmitted through contaminated infant formula, causing neonatal meningitis, sepsis, and necrotizing enterocolitis. The fatality rate can reach up to 80%.

This product is a freeze-dried real-time PCR reagent for the detection of Cronobacter. It uses the highly conserved ompA gene of Cronobacter as the detection target. It is a mastermix containing all necessary ingredients, except for template DNA, and it is pre-dispensed as single-test in PCR tubes for easy use. This product does not require cold chain for transportation and storage, which significantly reduces shipping cost, and eliminates possible loss by reagent wastage and aerosol contamination.

2. SPECIFICATIONS & COMPOSITION

3. STORAGE AND SHELF LIFE

Store at ambient temperature (5-30°C). It is stable for up to 12 months. Once the vacuum package is open, please store the unused products in the provided re-sealable plastic bag with the desiccants, and in the aluminum foil bag.

CAUTION:

The reagent pellet getting smaller is an indication of moisture seeping into the tube and the reagent is dampened. Any reagents with significant smaller pellet size than usual should be discarded or tested with positive control before use for sample testing.

4. ADDITIONAL EQUIPMENTS & REAGENTS REQUIRED

1) Real-time PCR instrument

2) Pipettes and tips

3) Nuclease-free water

4) Nucleic acid extraction kit

5. COMPATIBLE REAL-TIME PCR SYSTEMS

ABI 7500/Fast, Roche LightCycler 480II, BioRad CFX96, Bioer LineGene 9600.

6. ACCEPTABLE SPECIMENS

Food enrichment broth, vomitus, diarrheal specimens, etc.

7. OPERATION PROCEDURE

1) Nucleic Acid Extraction

Extract DNA from specimens using a proper extraction kit. It is recommended that the DNA is eluted with about 100μl of elution buffer (TE or nuclease-free H₂O) in the final step of extraction. The purified nucleic acid should be used immediately or stored at -20°C.

2) Positive Control Preparation

The positive control can be stored at ambient temperature before rehydration for up to 12 months. It should be rehydrated before use by adding 250 μl of TE buffer or nuclease-free H₂O, vortexed at low speed for 15-20 seconds, and centrifuged at low speed for 15-20 seconds. Use immediately or store at -20°C.

3) Real-Time PCR Mix Preparation

A) Open the vacuum package and take out the 8-tube strips containing the reagent. Check the pellet is at the bottom of the tube (Cut the number of tubes as needed if necessary). If the provided tubes are not compatible with your instrument, transfer the reagent pellet to an optical tube compatible with your instrument.

B) Open the tubes and discard the cap(s) (not suitable for real-time PCR machines), and prepare the reaction mix on ice as below.

step of extraction. The purified nucleic acid should be used immediately or stored at -20°C.

2) Positive Control Preparation

The positive control can be stored at ambient temperature before rehydration for up to 12 months. It should be rehydrated before use by adding 250 μl of TE buffer or nuclease-free H₂O, vortexed at low speed for 15-20 seconds, and centrifuged at low speed for 15-20 seconds. Use immediately or store at -20°C.

3) Real-Time PCR Mix Preparation

A) Open the vacuum package and take out the 8-tube strips containing the reagent. Check the pellet is at the bottom of the tube (Cut the number of tubes as needed if necessary). If the provided tubes are not compatible with your instrument, transfer the reagent pellet to an optical tube compatible with your instrument.

B) Open the tubes and discard the cap(s) (not suitable for real-time PCR machines), and prepare the reaction mix on ice as below.

* Nuclease-free water can be used as negative control.

• Cap the PCR using caps (strips) suitable for real time PCR(not provided).

D) Vortex the tubes at low speed for 10~15sec, and centrifuge at 3000rpm for 20sec and place them in a real-time PCR instrument.

2) RT-PCR Setup

Set the reaction volume as 25μl, and PCR amplification procedure as below. Collect FAM fluorescence at 60°C, and select NONE as passive reference.

3) Result Analysis and Interpretation