Listeria monocytogenes fluorescence real-time PCR test

Real-time fluorescent PCR in vitro detection of Listeria monocytogenes.

Specification: 96 T / box
The kit is stored at 2 - 8℃.
The validity period is 12 months.

Specification: 96 T / box

Uses: Real-time fluorescent PCR in vitro detection of Listeria monocytogenes.

Detection principle: The kit contains Listeria monocytogenes-specific primers. When the sample contains Listeria monocytogenes, the Listeria monocytogenes DNA extracted after pre-enrichment is exponentially amplified by PCR. During the reaction, the Listeria monocytogenes-specific fluorescent probe hybridizes with the target nucleic acid and is simultaneously Taq enzyme Hydrolysis to separate the fluorescent group from the quenching group on the probe: so that the presence of Listeria monocytogenes can be judged in real time by the fluorescence increment.

Product and composition:

Composition (96T/kit)	Specification	Quantity
DNA extraction solution	3ml	1 tube
Listeria monocytogenes PCR reaction solution	1100ul	1 tube
Tap enzyme (5U/ul)	20ul	1 tube
Listeria monocytogenes positive control	40ul	1 tube
Listeria monocytogenes Negative Control	0.5ml	1 tube
Instruction manual		1

Storage conditions and expiration date of the kit:
1. The kit is stored at 2 - 8℃.
2. The validity period is 12 months.

Applicable instruments:
ABI7500, 9600 series, MJ opticon2, Bio-Rad and other fluorescent quantitative PCR instruments are available

Experimental method:
1. Take 1ml of the enrichment solution into a 1.5m1 sterile centrifuge tube, centrifuge at 10.000rpm for 5min, and discard the supernatant; use the classic phenol chloroform extraction method to test the extracted template or store it at 20C until it is tested.
2. Place the PCR reaction solution at room temperature to equilibrate, take the Taq enzyme after melting, and add the Taq enzyme at 2∪/tube, that is, each test reaction system is prepared as: PCR reaction solution 22.5u1+0.4ulTaq enzyme.
3. Add the template: Thaw the nucleic acid extract stored at -20°C at room temperature and centrifuge at 13,000 rpm for 5 minutes. The negative and positive controls are thawed at room temperature before use. In each PCR reaction tube, add 2 ul of the template or negative and positive controls processed in step 1, cover the tube, place on the PCR instrument, and record the corresponding sample number.
4. On-board amplification and detection area: the reaction conditions are 95C: 3min, 1 cycle; 95C: 5sec, 60C: 40sec (signal acquisition), 40 cycles. The reaction system was set to 25u1.
For the multi-channel fluorescence PCR instrument, the signal is set to Fam fluorescein.

Results judgment:
Please refer to the manual for details.

Precautions for using the kit:
1. This kit is only used for in vitro testing and research purposes, please read the full text of this manual carefully before starting to use it.
2. The experiment must be strictly partitioned: pre-PCR preparation area-prepare experimental reagents; sample processing area-to be tested samples, template processing; detection area-PCR amplification, detection.
3. For the relevant laboratory management standards, please refer to the implementation of the management standards for gene amplification laboratories issued by relevant national authorities.

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Trivia questions : Listeria monocytogenes

About listeria

Listeria definition

Listeria is a facultative anaerobic bacterium that is the causative agent of listeriosis. Listeria is a Gram-positive bacterium belonging to the thick-walled phylum, named after Joseph Lister. It mainly uses food as a vector and is one of the deadliest foodborne pathogens.

Listeria is ubiquitous in the environment and can be found in most foods. Meat, eggs, poultry, seafood, dairy products, vegetables, etc. have all been proven to be sources of Listeria infection. Listeria monocytogenes poisoning can cause blood and brain tissue infections. Many countries have taken measures to control Listeria monocytogenes in food and have set corresponding standards.

Listeria sources

Listeria was first discovered in 1926 by a British scientist of South African origin Murray in a sick rabbit. In commemoration of the father of modern disinfection surgery, British physiologist Joseph Lister (1827-1912), in 1940, it was named Listeria monocytogenes at the 3rd International Congress of Microbiology.

Listeria species

The internationally recognized Listeria monocytogenes has the following seven strains:
Listeria monocytogenes, Listeria mutans, Listeria innocolus, Listeria welsii, Listeria sileeri, Listeria monocytogenes, Listeria monocytogenes. Among them, L. monocytogenes is the only one that can cause human diseases. It is a pathogen of zoonotic diseases. Severe poisoning can cause blood and brain tissue infections. Listeria monocytogenes is abbreviated as Listeria monocytogenes, which is a Gram-positive bacterium and belongs to the thick-walled phylum.

Listeria infection

In the genus Listeria, only Listeria monocytogenes can cause diseases such as meningitis, sepsis, miscarriage, and neonatal infection in humans. If an immunodeficiency person develops Listeria infection, the disease is serious and the mortality rate Up to 33%.

Listeria symptoms

Listeria infection can be accompanied by fever, neck pain, vomiting or diarrhea. More complex forms can also cause encephalitis and specific forms of meningitis. Pregnant women are also at risk of contracting Listeria. Pregnant women are 20 times more likely to be infected with Listeria than others. Even if there is no miscarriage or stillbirth, the newborn may have this infectious disease from birth, so there is a very high mortality rate.

Listeria monocytogenes

What are listeria monocytogenes

Listeria monocytogenes is a pathogen of zoonotic diseases. It can cause listeriosis in humans and animals. After infection, it is mainly manifested as sepsis, meningitis and mononuclear cells. Because the bacterium is an intracellular parasite, its removal by the host mainly depends on the cellular immune function. Therefore, listeriosis is mainly in neonates, the elderly, and immunocompromised people. Listeria monocytogenes is mainly infected through the feces, and can also enter the body through the eyes and damaged skin and mucous membranes to cause infection.

Distribution of Listeria monocytogenes

Listeria monocytogenes widely exists in nature, is not easy to be frozen and thawed, and can tolerate higher osmotic pressure. The bacteria are present in soil, surface water, sewage, wastewater, plants, silage, rotten vegetables, so animals can easily ingest the bacteria.

The transmission route of Listeria monocytogenes

Listeria monocytogenes mainly uses food as an infectious agent. It is one of the most deadly food-borne pathogens, causing 20 to 30% of infected people to die. The mortality rate is even higher than that of Salmonella and Botox. People mainly eat soft cheese, underheated chicken, unheated hot dogs, fresh milk, pasteurized milk, ice cream, raw steak, lamb chops, cabbage salad, celery, tomatoes, French pies, frozen pork tongue And so on. About 80% -90% of cases are caused by contaminated food.
The bacteria can enter the body through the eyes and damaged skin and mucous membranes to cause infection. After infection, pregnant women infect the fetus or newborn through the placenta or birth canal. The bacteria that inhabit the vagina and cervix can also cause infections. Sexual contact is also a possible route of transmission of the disease, and there is an upward trend.

Listeria monocytogenes disease

Listeria monocytogenes symptoms

Whether Listeria monocytogenes becomes ill after entering the human body is related to the virulence of the bacteria and the age and immune status of the host, because the bacterium is an intracellular parasitic bacterium, and its removal by the host mainly depends on the cellular immune function. Therefore, susceptible persons are newborns, pregnant women and adults over 40 years old. In addition, alcoholics, persons with damaged or defective immune systems, patients receiving immunosuppressive agents and corticosteroids, and organ transplants are also susceptible to infection by the bacteria. The clinical manifestations of the disease, healthy adult individuals have mild influenza-like symptoms, neonatal, pregnant women, immunodeficiency patients with shortness of breath, vomiting, hemorrhagic rash, suppurative conjunctivitis, fever, convulsions, coma, spontaneous abortion, meningitis, Sepsis until death.

Listeria monocytogenes treatment

Penicillin, ampicillin, gentamicin, erythromycin, etc. can be used for the treatment of Listeria monocytogenes infection. Erythromycin and other broad-spectrum antibiotics are allergic to penicillin.

Listeria monocytogenes prevention

Listeria monocytogenes can survive normal thermal processing, heat treatment has killed the competitive bacterial community, making Listeria monocytogenes easy to survive in the absence of competitive environmental conditions, so in food processing, the core temperature must reach 70 °C for more than 2 minutes. Listeria monocytogenes is widespread in nature, so even if the product has been overheated and processed, the Listeria monocytogenes is fully inactivated, but it may cause secondary pollution of the product, so after cooking, prevent secondary Pollution is extremely important. Because Listeria monocytogenes can still grow and reproduce at 4 °C, unheated refrigerator food increases the risk of food poisoning, and refrigerator food needs to be heated before consumption.

Listeria monocytogenes test

1. Bacterial culture method

The traditional test method for Listeria in food is to carry out pre-enrichment or selective enrichment, and use the isolated and cultured suspicious colonies for biochemical reaction experiments, hemolysis experiments, and cooperative hemolysis experiments (CAM P). After Listeria monocytogenes was further serotyped. Enrichment and selective enrichment are indispensable steps in this method. The methods of enrichment mainly include: cold enrichment method and normal temperature cultivation method. The cold enrichment method is cultured at 4° C for 30 days, sometimes even up to one year. At room temperature, the bacteria need to be cultured for 24h to 7d. Therefore, the traditional detection method requires 7-11 days to isolate and identify Listeria monocytogenes, so the traditional method detection period is longer.

2. Serum agglutination

According to bacterial antigens and flagellar antigens, LM is divided into 16 serotypes: 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 5, 6a, 6b, 7. Antigen structure has nothing to do with virulence. The main pathogenic factors for humans are serotypes 1/2a, 1/2b, and 4b, accounting for about 90% of global cases of this disease.

3. Molecular biology testing methods

3 .1 　 Probe detection technology
This technology is the earliest molecular organism used in the detection of Listeria
Learn technology. As early as 1988, Atin et al. cloned and sequenced a 500bp DNA fragment of the Listeria beta-hemolysin gene, and synthesized four oligonucleotide probes from this sequence, labeled the probe with 32P, and performed a dot hybridization experiment It was confirmed that the probe only reacted with Listeria monocytogenes, and it was negative with other species of Listeria and non-genus bacteria, showing good specificity.

3.2 Polymerase chain reaction (PCR) detection technology
The development of PCR is quite mature, and it is widely used in the detection of Listeria. PCR amplification specific primers are designed based on the specific virulence gene sequence of Listeria monocytogenes, and one is designed based on the specific sequence in the genome of Listeria monocytogenes. Commonly used target sequences are virulence genes such as hly A, iap, inl, Dth-18. The advantage of using the PCR method to detect Listeria is the specificity of the method, but the disadvantage is the low sensitivity; the sample culture is amplified after chemical extraction, which improves the detection rate of the sample; and can A "live, non-cultivable" Listeria monocytogenes could not be detected by traditional enrichment methods.