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Enterobacter sakazakii fluorescence real-time PCR test

salmonella

Real-time fluorescent PCR in vitro detection of enterobacter sakazakii.

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 enterobacter sakazakii.

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

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

About enterobacter sakazakii

What is enterobacter sakazakii

Enterobacter sakazakii (ES) is a Gram-negative non-Bacillus parasitic in the intestines of humans and animals, belonging to the family Enterobacteriaceae. They can cause disease to humans and animals under certain conditions, so they are called conditional pathogens.

The hazards of enterobacter sakazakii

Enterobacter sakazakii can cause severe neonatal meningitis, enterocolitis, bacteremia, and neurological dysfunction. In 1961, the United Kingdom first reported two cases of meningitis caused by Enterobacter sakazakii. Later, the United States, the Netherlands, and Canada and other countries successively reported neonatal Enterobacter sakazakii infections. Infants infected with Enterobacter sakazakii have a mortality rate as high as 20%-50%, and surviving infants and young children may also have severe neurological sequelae.

Enterobacter sakazakii disease

In 1961, two scientists, Urmenyi and Franklin in the UK, reported for the first time two cases of meningitis caused by the bacterium, followed by meningitis, septicemia, and necrotizing small intestine colon caused by Enterobacter sakazakii in succession worldwide reports.

Enterobacter sakazakii infection in the newborn

Cases of sporadic and outbreaks of meningitis, septicemia, and necrotizing colitis caused by Enterobacter sakazakii in infants and premature babies have appeared one after another in the world. Several studies have shown that infant formula is currently the main channel of infection found to cause meningitis, sepsis and necrotizing colitis in infants and preterm infants. In some cases, mortality caused by disease caused by Enterobacter sakazakii can reach 40%~80%. Enterobacter sakazakii has attracted the attention of relevant departments in many countries around the world.

Enterobacter sakazakii symptoms

Most children have mild and atypical clinical symptoms and are easily overlooked. Severe cases can cause necrotizing enterocolitis, sepsis, and meningitis.
(1) Symptoms of the system: Fever. Newborns can show signs of hypothermia, mental depression, milk rejection, increased jaundice, gray complexion, flaky skin and even shock.
(B) Symptoms of the digestive system: vomiting, abdominal distension, diarrhea, mucus bloody stools, bowel sounds weaken or even disappear, in severe cases, bowel perforation and peritonitis can occur.
(3) Symptoms of the nervous system: irritability, sharp crying, drowsiness and even coma, gaze, convulsions may appear, physical examination may include enlarged head circumference, split craniotomy, increased bregma tension, and positive meningeal irritation signs.

Enterobacter sakazakii treatment

(1) Mild cases should be strengthened and treated symptomatically to ensure the intake of fluids and electrolytes, and pay attention to follow-up.
(2) Those who have obvious intestinal symptoms should choose antibacterial drugs. Amoxicillin/clavulanic acid and other antibacterial drugs can be selected and adjusted according to the results of drug susceptibility tests. Intestinal mucosal protective agents and microecological agents can be used at the same time.
(3) In severe cases, ceftazidime, cefepime, melopenem, etc. can be used for antibacterial treatment, and adjusted according to the clinical treatment effect and drug sensitivity test results. Symptomatic support treatment should be strengthened; children with necrotizing enterocolitis should be fasted, gastrointestinal decompression should be given to those with severe abdominal distension, and surgical treatment should be given if necessary; children with meningitis should be given sedation, anxiety reduction, intracranial pressure reduction and complications Treatment; give anti-shock treatment in time when there is shock.

How is enterobacter sakazakii transmitted

Enterobacter sakazakii is Enterobacteriaceae, Gram-negative bacilli. Enterobacter sakazakii is widely distributed in nature and reproduces rapidly, but it is not heat-resistant and can be killed by pasteurization. Enterobacter sakazakii is a conditionally pathogenic bacteria. Under normal circumstances, it does not cause harm to human health, but it can cause disease for people with low immunity and infants, newborns, especially premature infants and low-birth-weight infants.

Enterobacter sakazakii common food sources

Food and environment are the main sources of E. sakazakii contamination. Scientists have isolated enterobacter sakazakii from a variety of foods and beverages. Various raw materials of these foods and beverages, such as milk powder, meat products, soy products, bread, etc., may be infected with enterobacter sakazakii, resulting in the detection of the bacteria in the final product. Among them, infant formula milk powder is called the most common and most sensitive food source of enterobacter sakazakii.

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.

Enterobacter sakazakii prevention

For the contamination of Enterobacter sakazakii in infant foods, the contamination of the bacteria should be controlled from the source, processing and packaging materials of infant foods. First of all, we should strengthen the pollution prevention and control of food ingredients for infants and young children. Strict disinfection and inspection of raw materials should be carried out. The contaminated raw materials should be processed in time. Do not enter the production workshop to avoid pollution to other raw materials.

Secondly, strictly sterilize all links in the processing process, such as the operator's mouth, hands, feet, head, shoes, work clothes, caps, etc., must be done hygienic work. At the same time, the bacteria are completely killed by pasteurization, ultra high temperature sterilization or other high temperature processes during the processing.

To prevent E. sakazakii contamination, an effective hazard analysis and quality control system for critical control points should also be established and strictly implemented during the production process. In addition, the packaging materials should be effectively disinfected, and the entire workshop environment for production and packaging should be disinfected regularly.

In addition, in the feeding of infants and young children, since there is also a risk of infection of Enterobacter sakazakii in the family, it is recommended that bottles and other feeding utensils for milk powder be thoroughly cleaned and disinfected. In addition, breastfeeding is also one of the effective means to reduce the risk of neonatal infection with E. sakazakii.

Enterobacter sakazakii biochemical identification

Traditional inspection methods

The traditional method of separation and detection of Enterobacter sakazakii depends on biochemical and morphological characteristics. Internationally, "Separation and Counting of Enterobacter sakazakii in infant formula" promulgated by the US FDA is usually used as the classic method.

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.

Molecular biology testing methods

At present, the molecular biological testing methods of E. sakazakii used in dairy products include ordinary PCR method (electrophoresis method), DNA loop-mediated constant amplification (LAMP) testing method and fluorescent PCR method. This method overcomes the shortcomings of traditional inspection methods such as cumbersome operation, time-consuming, and difficult to distinguish results. It has also led to the rapid development of rapid detection methods for Enterobacter sakazakii. However, this method also has the disadvantages of high concentration of bacteria to be measured in the sample, high analysis cost, cumbersome operation, and inability to identify the presence of live bacteria.

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