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Salmonella Fluorescence real-time PCR test

salmonella

Real-time fluorescent PCR in vitro detection of Salmonella.

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 Salmonella.

Detection principle: The kit contains Salmonella-specific primers. When the sample contains Salmonella, the Salmonella DNA extracted after pre-enrichment is exponentially amplified by PCR. During the reaction, the Salmonella-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 Salmonella can be judged in real time by the fluorescence increment.

Product and composition:
Composition (96T/kit)SpecificationQuantity
DNA extraction solution3ml1 tube
Salmonella PCR reaction solution1100ul1 tube
Tap enzyme (5U/ul)20ul1 tube
Salmonella positive control40ul1 tube
Salmonella Negative Control0.5ml1 tube
Instruction manual1
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 : Salmonella

About Salmonella

Salmonella definition

Salmonella is a general term for a group of bacteria. It can infect humans and cause diseases as well as animals. It is a zoonotic disease. Because Salmonella is a Gram-negative Enterobacteriaceae, people can eat food contaminated with Salmonella and cause food poisoning. According to statistics, in the world of food poisoning, mainly caused by the Salmonella diet is a common food-borne pathogen. In addition, Salmonella typhimurium, Salmonella paratyphi, Salmonella choleraesuis, etc., can even cause intestinal epidemics such as typhoid fever, paratyphoid fever, and cholera. Typhoid fever only infects humans, not animals, and damages human bone marrow, liver and spleen. Every year, it can cause tens of millions of people to get sick and more than 600,000 to die.

Salmonella sources

Salmonella has a wide range of hosts in nature. A few Salmonella are selective for the host, and most of them are suitable for humans and animals. They can live in the gastrointestinal tract of mammals, reptiles, birds, insects, and humans. The infection rate with wild animals is more than 1% to 20%.

Salmonella mainly contaminates meat food, and fish, poultry, milk, and egg foods can also be contaminated by these bacteria. Salmonella food poisoning can occur throughout the year. Eating uncooked disease, dead livestock meat, or livestock meat contaminated at other points after slaughter is the most important cause of salmonella food poisoning. Some food experts point out that Americans have different eating habits from Chinese people. They like to eat half-boiled eggs or even raw eggs, so once the eggs contain salmonella, the chance of infection is relatively high. "In the country, it is not surprising that raw eggs contain salmonella, but people like to cook eggs cooked, which greatly reduces the chance of infection.

Salmonella infection

Salmonella belongs to the Enterobacteriaceae family and can cause gastroenteritis, typhoid fever, sepsis, and extraintestinal focal infections. It is collectively referred to as Salmonella infection (ie, salmonellosis). Various syndromes can sometimes overlap. Infections caused by typhoid and paratyphoid bacteria are not included.

The clinical manifestations of Salmonella infections are diverse. According to their main symptoms, they can be divided into four types: enteritis, typhoid, septicemia, and localized suppurative infections.

Salmonella species

Salmonella typhi

Salmonella typhi is a bacterium with a size of (0.6 to 0.9) × (1 to 3) microns without spores and generally no capsule. Except for Salmonella gallinarum and Salmonella typhi, most of them have flagella around the body, causing Salmonella typhi.
Salmonella bacteria that cause typhoid fever. Bacteria are rod-shaped, with flagella all over the body and can produce endotoxin. Endotoxin injection into rabbit veins can cause human-like lesions, such as fever, leukopenia, etc., and massive injections can cause death.

Salmonella typhimurium

Salmonella typhimurium is a group of non-adaptive or pantrophic salmonella. It has a wide range of hosts and is distributed in nature. It exists in the intestinal tract of various animals such as poultry, livestock, and rodents. One. The bacteria can cause various infectious diseases of poultry and mammals, and can also cause human infections, and has important public health significance.

Salmonella enteritidis

Salmonella enteritidis is the main pathogen causing acute gastroenteritis. Typical symptoms after infection include fever, diarrhea, and vomiting. Salmonella enteritidis is one of the pathogenic bacteria that has no host specificity and is invasive. The host includes humans and various animals. Not only can the bacteria cause serious economic losses to the morbidity and mortality of poultry, but the contaminated poultry products as carriers of Salmonella enteritidis also seriously endanger human health.

Salmonella choleraesuis

Salmonella choleraesuis is a type of non-typhoidal Salmonella, which is the main pathogenic bacteria causing paratyphoid in piglets, causing great harm to the pig industry.
When the septicaemic Salmonella choleraesuis invades the body of the pig, it immediately invades the lymphatic space of the intestinal wall, thereby invading the blood again and multiplying in the blood, causing septicemia. A large number of endotoxins are produced when the bacteria reproduce, and endotoxins act on white blood cells and cause inflammation, high fever and diarrhea.
The infection of Salmonella choleraesuis is mainly transmitted through the faecal orifice. If the patient has a pig farm in the home, the hygiene habits are poor, and the basis of chronic liver disease is low, the immunity is low, so it may be due to the intake of food or water contaminated with Salmonella Cause infection.

After adult infection with Salmonella choleraesuis, the onset is insidious and the clinical manifestations are not typical. It is easy to miss diagnosis and misdiagnosis and delay treatment. If the medication course is insufficient, the insidious bacteremia control is incomplete and the condition can be prolonged repeatedly.

Salmonella poisoning

The host specificity of Salmonella is very weak, which can infect both animals and humans, and it is easy to cause food poisoning in humans. The most pathogenic is Salmonella cholerae, followed by Salmonella typhimurium and Salmonellaenteritidis.
Ingestion of non-typhoidal Salmonella contaminated food by mouth caused illness. Common foods that cause illness are mainly animal foods, mainly livestock meat, poultry meat, eggs, milk and their products.

Salmonella symptoms

Symptoms of food poisoning caused by salmonella mainly include nausea, vomiting, abdominal pain, headache, chills and diarrhea, as well as fatigue, muscle aches, blurred vision, moderate fever, restlessness and drowsiness, the duration lasts 2 to 3 days, average the lethal rate was 4.1%. The main reason is caused by the ingestion of foods containing large amounts of non-host specific bacteria or serotypes of Salmonella.

Salmonella pathogenesis

1. The invasion of live salmonella bacteria on the intestinal mucosa and the synergistic effect of endotoxin; it is the main cause of food poisoning caused by salmonella.

2. Enterotoxin poisoning: Salmonella enteritidis and Salmonella typhi can produce enterotoxin. The mechanism is the same as the enterotoxin type of bacterial food poisoning.

Salmonella incubation period

After ingesting toxic food. Symptoms generally appear within 12 to 14 hours, and some have a longer incubation period.

Salmonella treatment

Gastroenteritis caused by Salmonella generally does not require antibiotic treatment; it is mainly supplemented with water and electrolytes; but for neonates, immunocompromised patients and special wounded patients; antibiotic treatment is required depending on the situation.

Salmonella prevention

1. Wash your hands carefully before eating, after going to the toilet, before touching food, after touching animals or raw eggs.
2. Separate the cutting boards for raw and cooked food.
3. Eat the food thoroughly (especially eggs and poultry).
4. Food that is not ready-to-eat should be wrapped in plastic wrap and stored in the refrigerator. It should be heated or cooked before being eaten again.
5. Fight and block vectors such as flies. Unclean food contaminated with flies, expired or spoiled should be discarded and not eaten.
6. The water tower should be cleaned and disinfected frequently.
7. Drinking water during travel or camping should be boiled and disinfected.
8. If you have symptoms such as vomiting, diarrhea or fever, you should seek medical treatment as soon as possible.

Salmonella test

Antibody detection

The significant specificity of antigen-antibody reaction has been used for bacterial identification and serological typing for more than half a century. The presence of specific antibodies to bacterial cells or flagellar antigens allows people to establish rapid methods to detect foodborne pathogens. There are many established immunological detection methods for Salmonella, which can be roughly divided into methods based on enzyme-labeled antibodies (ELISA), fluorescent antibody staining (immunofluorescence), isotope-labeled antibodies (radioimmunoassay), and many other methods. Antibody-based methods using latex agglutination, immunosensors, immunodiffusion and immunochromatography. However, the most widely used method is based on the two-site ELISA technique, namely sandwich ELISA. After the improvement of this method, radioactive isotopes are used to replace labeled antibodies. In a nutshell, it refers to the use of "capture" antibodies immobilized on a solid substrate to capture the target antigen. After washing to remove unbound components, a second enzyme label is added Antibodies, which are bound to different sites of the captured antigen, are added to the enzyme substrate after the second wash, and allowed to react with the color components, and then the target antigen can be easily detected by spectrophotometry. Using microtiter plates as a solid matrix standardizes the reaction format and facilitates its automation.

Nucleic acid method

Cellular nucleic acid DNA and RNA are the only large molecules that can carry information. Since all cells contain this molecule, it can be used as a target for detection. The target is usually a specific nucleic acid sequence, which can be detected by using complement nucleic acid molecules as probes. Similar to immunological methods, the probe also needs to be appropriately labeled, such as radioisotopes, enzymes or luminescent markers. Fitts et al. introduced the first generation of DNA-RNA hybridization technology in the detection of Salmonella in food. The probes used in this method contain DNA fragments of Salmonella typhi which are labeled with radioactive isotopes. Their sensitivity is high. After about 48h of the enrichment step, the detection limit can reach 108 bacteria/ml, but due to the use of radioisotopes, which can only be applied in specialized laboratories, the advantages of this method are offset. To this end, a second-generation technology based on nucleic acid hybridization, a colorimeter, has been developed.

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