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E coli fluorescence real-time PCR test


Real-time fluorescent PCR in vitro detection of E coli.

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 e coli.

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

Product and composition:
Composition (96T/kit)SpecificationQuantity
DNA extraction solution3ml1 tube
E coli PCR reaction solution1100ul1 tube
Tap enzyme (5U/ul)20ul1 tube
E coli positive control40ul1 tube
E coli 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 : E coli

About e coli

E coli, also known as Escherichia coli, was discovered by Escherich in 1885. Escherichia coli is a conditionally pathogenic bacteria. Under certain conditions, it can cause gastrointestinal infections or urinary tract infections of various tissues and organs in humans and various animals.

E coli size

The size is 0.5×1~3 microns. Chow flagella, can move without du spores. It can ferment a variety of sugars to produce acid and gas. It is a normal resident bacterium in the intestines of humans and animals. It enters the intestines with infants after birth and is accompanied by humans for life, accounting for almost 1/3 of the dry weight of feces.

E coli shape

E coli is Brevibacterium, with blunt round ends and Gram negative. Sometimes, due to different environments, individual cells appear to be spherical or filament-like; E. coli are mostly single or two, but they will not be arranged in a long chain shape; most E. coli strains have capsules or microcapsules Structure, but cannot form spores; most E. coli strains grow fimbriae, some of which are host-specific fimbriae that have adhesion to the host and some other tissues or cells.

E coli classification

Pathogenic effect
At present, the internationally recognized classification mainly includes six types of E. coli, namely intestinal pathogenic E. coli (EPEC), intestinal toxigenic E. coli (ETEC), and intestinal invasion that can cause gastrointestinal infections. E coli (EIEC), intestinal hemorrhagic E coli (EHEC), intestinal aggregative E coli (EAEC) and intestinal Shiga-like toxins found in recent years also have certain invasive E coli (ESIES) In addition, there are urethral pathogenic E coli (UPEC) that can cause urinary tract infection, and the newly named intestinal agglomerative adherent E. coli (EAggEC).

According to the ability of E. coli to produce hemolysin and the ability to hemolyze, E. coli can be divided into two categories: hemolytic E. coli and non-hemolytic E. coli.

According to the ability of E coli to produce enterotoxin during the infection process, E coli can be divided into two categories: enterotoxigenic E coli and non-entotoxinogenic E. coli. Enterotoxigenic E. coli is an important pathogen of any infectious diarrhea in humans and various animals. The identification of enterotoxigenic E. coli is mainly to determine the type of enterotoxin secreted by the isolated E. coli. In addition, according to the ability of E. coli to produce enterotoxin, combined with its sensitivity to different enterotoxins, E coli can be classified as enterotoxin type.

E coli infection

E. coli is normal resident bacteria in the intestines of animals, and a small part of them cause disease under certain conditions. The serotypes of E. coli can cause gastrointestinal infections in humans or animals, mainly caused by specific fimbria antigens, pathogenic toxins and other infections. In addition to gastrointestinal infections, they can also cause urinary tract infections, arthritis, and meninges. Inflammation and septic infections.
Escherichia coli includes several bacterial groups with different pathogenicity. Human E. coli infections are roughly divided into two types: intestinal infections and extraintestinal infections.

E coli symptoms

The most typical symptom of intestinal infection may be diarrhea. If the infection is severe, shock may occur, blood pressure may drop, and heart rate may increase. If it is an intestinal infection, nausea, vomiting, fever, urinary frequency, and urgency may occur after E. coli infection.

E coli pathogen

Generally, E. coli is divided into four bacterial groups according to its pathogenicity to humans: common E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, and invasive E. coli. The latter three are pathogenic bacteria that cause diarrhea.

E coli pathogenesis

Each flora includes a certain number of serotypes. Enterotoxigenic Escherichia coli can produce heat-resistant enterotoxin and heat-resistant enterotoxin. The production of these two enterotoxins is genetically controlled by plasmids. The plasmid is a kind of non-chromosomal DNA that can be easily transmitted. The mutual transmission of plasmids can enable the non-toxin-producing strains to obtain toxin-producing ability. Due to the delivery of the plasmid, Escherichia coli of any serotype can produce enterotoxin and colony factor, thus having pathogenicity.

E coli treatment

First, if the symptoms of infection are not very serious and the diarrhea and abdominal pain are mild, oral administration of bifidobacteria can improve gastrointestinal dysfunction and regulate intestinal microflora disorders.
If the abdominal pain and diarrhea are serious and there is watery stool, consider giving anti-inflammatory treatment such as quinolone antibiotics or berberine, and appropriate fluid replacement to improve dehydration symptoms.

Where does e coli come from

Escherichia coli is one of the most common microorganisms, widely distributed in nature, most of which are not pathogenic, mainly parasitic in the intestines of humans or animals, and are called "normal flora" by humans.

E coli transmission

The main source of infection of human E. coli disease is that a large number of E. coli pathogens are excreted in the feces of gastrointestinal infection patients. The transmission route of E. coli among people is mostly through the fecal-oral transmission route, which can cause the spread or epidemic of E. coli disease under certain conditions.

Enterobacter sakazakii in powdered infant formula

Enterobacter sakazakii is a creature that exists in the environment and is commonly found in facilities and home environments where infant formula is produced. Although infant formula is not a sterile product and may be contaminated with pathogens that can cause serious diseases, if the infant formula is properly prepared and stored, the risk of infection by Enterobacter sakazakii can be greatly reduced. To safely prepare infant formula, you must first clean and sterilize the feeding and preparation equipment.

E coli prevention

1. Keep the bai place and kitchen utensils clean, and properly dispose of garbage.
2. Keep your hands clean and trim your nails frequently.
3. Wash your hands with soap and water before eating or handling food. You should also wash your hands after using the toilet or changing diapers.
4. Tap water should be used for drinking water, and it is best to boil it before drinking.
5. Fresh food should be purchased from a reliable place, not patronizing unlicensed hawkers.
6. Avoid eating high-risk foods, such as milk that has not been processed by low-temperature sterilization, as well as undercooked hamburger steaks, ground beef, and other meat foods.
7. When cooking food, wear clean, washable apron and a hat.
8. Food should be thoroughly washed.
9. Perishable food should be covered with a lid and stored in the refrigerator.
10. Raw food and cooked food, especially beef and beef offal, should be handled and stored separately (store cooked food on the top of the refrigerator and raw food on the bottom) to avoid cross-contamination.
11. The refrigerator should be cleaned and melted regularly, and the temperature should be kept at 4 degrees Celsius or below.
12. If all parts of the food are heated to 75 degrees Celsius, E. coli O157:H7 can be eliminated; therefore, ground beef and hamburger steak should be thoroughly cooked to 75 degrees Celsius for 2 to 3 minutes, until the cooked meat is completely turned It is brown, and the gravy becomes clear.
13. Do not handle cooked food with bare hands; if necessary, wear gloves.
14. Food should be consumed as soon as possible after cooking.
15. If it is necessary to keep leftover cooked food, it should be refrigerated and consumed as soon as possible. Reheat thoroughly before eating. Spoiled food should be discarded.

Test for e coli

Fermentation method

This method is mainly for the cultivation of E. coli on a medium at 44.5°C, which contains a fluorescent substrate and requires 24 hours of cultivation. The main steps include fermentation, separation culture, secondary fermentation, microscope observation, etc.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MAL-DITOF-MS) method is to mix the sample to be measured with the matrix solution at a certain ratio, and use pulsed laser irradiation to excite the matrix, causing the polymer to generate molecular ions and molecular ion polymerization The body is accelerated and detected in a time-of-flight mass spectrometer. As a result, the measured polymer is separated according to the mass number, processed to form a mass spectrum, and analyzed and compared by software to screen and determine the specificity (fingerprint) spectrum to achieve Differentiation and identification of target microorganism species or strains.

Immunomagnetic bead method

The main principle of the separation technology is to use magnetic beads as a carrier and an antibody to combine the antibody and the magnetic beads, and then to complete the mechanical movement through magnetic technology to separate E. coli. Compared with other ways of separating bacteria, this method has certain advantages. This technology can improve the detection success rate of pathogenic Vibrio in samples, and the immunomagnetic bead technology can treat different microorganisms in different bacterial species. , And to a large extent improve the detection efficiency.

Automated instrument testing

The main use of immune automated analyzer, this technology was produced and used in 1970. With the development and progress of science and technology, automatic instrument detection technology is widely used, and it is very convenient to operate, it can save a lot of time, its degree of interference is small, it can save manpower and material input, and can also improve the accuracy of detection. In the current development process, the application of automatic enzyme immunoassay system is very extensive.

ATP bioluminescence

In the development process in recent years, bioluminescence technology is widely used, and it is a relatively rapid technology for detecting microorganisms. In active cells, ATP is its common energy metabolic product, which can provide the energy required during the physiological activities of cells. And, this technology can maintain a certain content within a certain range in the living body. The detection technology of Escherichia coli in food can adopt the method of fluorescence photometry, because the reason that the organism emits light is the role of luciferase, which produces the effect of light emission. The substance comes from the fireflies in North America and can catalyze the oxidation of fluorescein. However, the substance is unstable and can quickly decompose the fluorescence. In addition, the detection technology result acquisition process is very fast, and the device is easy to carry, very suitable for on-site detection.

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