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Listeria monocytogenes fluorescence real-time PCR test


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

Kev qhia tshwj xeeb: 96 T / lub thawv
Cov khoom siv tau khaws cia ntawm 2 - 8 ℃.
Lub sijhawm siv tau yog 12 lub hlis.
specification: 96T / lub thawv

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

Txoj cai tshawb nrhiav: 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.

Khoom thiab muaj pes tsawg leeg:
Muaj pes tsawg leeg (96T/khoom siv)specificationkom muaj nuj nqis
Kev daws cov tshuaj DNA3ml1 raj
Listeria monocytogenes PCR reaction solution1100ul1 raj
Kais enzyme (5U/ul)20ul1 raj
Listeria monocytogenes positive control40ul1 raj
Listeria monocytogenes Negative Control0.5ml1 raj
phau ntawv qhia1
Cov xwm txheej cia thiab hnub tas sij hawm ntawm cov khoom siv:
1. Cov khoom siv tau khaws cia ntawm 2 - 8 ℃.
2. Lub sijhawm siv tau yog 12 lub hlis.

Cov cuab yeej siv tau:
ABI7500, 9600 series, MJ opticon2, Bio-Rad thiab lwm yam fluorescent ntau qhov ntsuas PCR muaj

Txoj kev sim:
1. Siv 1ml ntawm cov tshuaj ua kom zoo rau hauv 1.5m1 cov menyuam tsis muaj menyuam centrifuge raj, centrifuge ntawm 10.000rpm rau 5min, thiab pov tseg cov supernatant; siv cov txheej txheem rho tawm phenol chloroform qub los kuaj cov ntawv rho tawm lossis khaws nws ntawm 20C kom txog thaum nws raug sim.
2. Muab PCR cov tshuaj tiv thaiv ntawm chav sov kom sib npaug, coj Taq enzyme tom qab yaj, thiab ntxiv Taq enzyme ntawm 2∪/raj, uas yog, txhua qhov kev sim tshuaj tiv thaiv kab mob tau npaj ua: PCR cov tshuaj tiv thaiv tshuaj 22.5u1+0.4ulTaq enzyme.
3. Ntxiv tus qauv: Thaw cov nucleic acid extract khaws cia ntawm -20 ° C ntawm chav sov thiab centrifuge ntawm 13,000 rpm rau 5 feeb. Qhov tsis zoo thiab kev tswj hwm tau yaj hauv chav sov ua ntej siv. Hauv txhua lub PCR cov tshuaj tiv thaiv raj, ntxiv 2 ul ntawm tus qauv lossis qhov tsis zoo thiab kev tswj hwm tau ua tiav hauv qib 1, npog lub raj, tso rau ntawm PCR ntsuas, thiab sau tus lej piv txwv.
4. On-board amplification thiab nrhiav chaw: qhov xwm txheej tshwm sim yog 95C: 3min, 1 lub voj voog; 95C: 5sec, 60C: 40sec (teeb liab tau txais), 40 mus. Cov tshuaj tiv thaiv kab mob tau teeb tsa rau 25u1.
Rau ntau-channel fluorescence PCR ntsuas, lub teeb liab tau teeb tsa rau Fam fluorescein.

Cov txiaj ntsig kev txiav txim:
Thov mus saib phau ntawv qhia kom ntxaws.

Ceev faj rau kev siv cov khoom siv:
1. Cov khoom siv no tsuas yog siv rau hauv vitro kuaj thiab tshawb fawb lub hom phiaj, thov nyeem tag nrho cov ntawv ntawm phau ntawv no ua tib zoo ua ntej pib siv nws.
2. Kev sim yuav tsum tau muab faib nruj me ntsis: thaj tsam npaj ua ntej PCR-npaj cov tshuaj reagents; cheeb tsam ua qauv-yuav tsum kuaj cov qauv, ua qauv; thaj tsam nrhiav pom-PCR kev nthuav dav, nrhiav pom.
3. Rau cov txheej txheem kev tswj hwm hauv chaw sim, thov saib mus rau qhov ua tiav ntawm cov qauv kev tswj hwm rau kev hloov pauv cov noob tso tawm los ntawm cov koom haum hauv tebchaws.

Yog tias koj xav paub ntau ntxiv, thov hu rau peb

    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.

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