Listeria qPCR kit

Clinical significance
Listeria monocytogenes is one of the most common food-borne pathogens, causes listeriosis. An estimated 1,600 people get listeriosis each year, and about 260 dies. The infection is most likely to sicken pregnant women and their newborns, adults aged 65 or older, and people with weakened immune systems. Listeria can survive and multiply at temperature as low as 0°C, but can be easily destroyed under normal cooking temperature. Pathogenic Listeria ingested in food survive passage through the gastric acid barrier and invade the mucosa of the small and large intestine and produce pore-forming toxins. The cholesterol-dependent cytotoxin Listeriolysin O is a major L. monocytogenes virulence factor that mediates the escape of bacteria from internalization vacuoles, allowing replication in the cytosol. The Hly gene, coding toxin Listeriolysin O is the main target for detection by kit «Listeria qPCR kit».

Principle of analysis
The method is based on the isolation of total DNA from pre-enrichment culture samples, and further amplification of specific regions of Listeria DNA (Hly gene) using specific oligonucleotide primers and the synthesis of complementary DNA chains using the enzyme Hot start Taq-DNA polymerase. Fluorescent detection of the analysis results is performed both in real-time (Real-time mode) or end point assay using two fluorescence detection channels (FAM525 nm, ROX580 nm), see table below:

Marker	Detection channel
Listeria, Hly gene	FAM
Internal control, 16S gene	ROX

Kit contents

Component name	Quantity
Listeria Master Mix (Lyophilized)	1 vial
Listeria Positive Control (Lyophilized)	1 tube

Storage and stability
The test kit reagents are stable for the shelf life indicated on the label (at least 12 months from manufacture date) if stored at room temperature.

Sample preparation
Foods, feeds and environment samples should be processed within 24 h of sampling following ISO 6579:2002 and ISO 7218:2007 procedures. After pre-enrichment 0,1-1,0 mL samples should be processed for nucleic acid extractions using commercially available DNA extraction kits. We recommend using our Nucleic acids extraction kit compatible with automatic Nucleic extraction device LTEXT1.

Reagent preparation
Master Mix preparation: Add distilled water 0,5 mL directly to Lyophilized Master Mix, wait 5 min, mixing well and dispense 5 µL into PCR testing tubes. Positive control preparation: Add distilled water 0,5 mL directly to Lyophilized Positive Control, wait 5 min, mixing well and use directly 5 µL for PCR or use 100 µL for Nucleic acid extraction (to control Nucleic acid extraction efficiency). The rest of TE buffer can be used as Negative Control. Reagents and controls must be kept in a chilled stand. After use reagents and controls can be freeze at minus 20°C for storage. Several freezing-thawing cycles are allowed.

Assay procedure
1. Select the required amount of the PCR tubes or wells in the PCR plate.
2. Using pipette tips with an aerosol filter dispense Master mix 5 µL add 5 µL of DNA samples (Positive and Negative control samples).
3. Place testing tubes in the fluorescence PCR instrument, and set the negative/positive control and unknown samples parameters for PCR reaction according to the instrument operating instructions. Fluorescence is measured at 60°C on the FAM, and ROX channels. The PCR parameters are shown in table below:

Step	Temp	Time	Repeats	Data collections
1	37 C	10 min	1	No
2	95 C	5 min	1	No
3	95 C	10 sec	45	No
3	60 C	30 sec	45	Yes

4. Start run.

Result records and their interpretation is shown in the table below:

Marker	Fluorescent channels	Salmonella Positive	Salmonella Negative
Listeria Hly gene	FAM	Ct≤ 45	Undetectable
Internal control gene 16S	ROX	Ct≤ 35	Ct≤ 35

Product specification:

Detection Method	Primer-probe
Format	Lyophilized in Glass vials
Reaction Speed	Fast
Disease	Listeriosis
Label or Dye
Target	FAM
Internal control	ROX
Target Organism Class	Listeria monocytogenes
Shipping Condition	Room temperature
For Use With (Application)	Pathogen detection
Concentration	2x
Validation	Field strains: L. monocytogenes Reference DNA: L. monocytogenes (BNCC363862)