Kit and application thereof as well as detection method

A kit, the technology of Sakazaki Krono, applied in the food field, can solve the problems of inability to distinguish dead bacteria from live bacteria and interfere with the authenticity of detection, so as to achieve the effect of improving specificity and avoiding false positive rate

Inactive Publication Date: 2015-08-26
贝因美股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most Cronobacter sakazakii have died after pasteurization in the production of infant formula milk powder. Because DNA can remain in dead bacteria for a long time, and the traditional fluorescent quantitative PCR method cannot distinguish dead bacteria from live bacteria. Interfering with the authenticity of the detection

Method used

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  • Kit and application thereof as well as detection method
  • Kit and application thereof as well as detection method
  • Kit and application thereof as well as detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Materials:

[0047] EMA was purchased from Biotium Company of the United States, Premix Ex Taq TM (2×) was purchased from TaKaRa Company, and 7500Fast quantitative PCR instrument was a product of Life Technology Company of the United States.

[0048] 2. Primer and probe design and synthesis:

[0049] Using the conserved gene sequence from the 3' end of the rpsU gene to the 5' end of the dnaG gene of Cronobacter sakazakii as a template, use Primer Express TM (V3.0, American AB Company) software analyzes TaqMan-MGB primers and probe sites, and selects the best combination therefrom.

[0050] Upstream primer: 5′-gca caa gcg gtg gag cat-3′

[0051] Downstream primer: 5′-aac cca aca ttt cac aac acg ag-3′

[0052] Fluorescent probe: 5′-FAM-ctt acc tgg tct tga cat c-MGB-3′

[0053] Wherein FAM is a fluorescent reporter group, and MGB is a fluorescent quencher group.

Embodiment 2

[0054] Example 2: Feasibility evaluation of real-time fluorescent PCR method for live Cronobacter sakazakii in infant formula milk powder

[0055] Take the concentration as 5.5×10 respectively 7 cfu / ml, 5.5×10 6 cfu / ml, 5.5×10 5 cfu / ml, 5.5×10 4 cfu / ml, 5.5×10 3 cfu / ml, 5.5×10 2 cfu / ml, 5.5×10cfu / ml 7 concentrations of Cronobacter sakazakii cultures were subjected to EMA real-time fluorescent PCR amplification according to the kit provided in Example 1.

[0056] Total reaction system: 25 μL total reaction system, including 2×Premix Ex Taq TM 12.5 μL, 0.3 μL each of upstream and downstream primers (20 μmol / L), 0.3 μL of probe (20 μmol / L), 0.2 μL of ROX, 5.0 μL of template DNA each, with ddH 2 O make up.

[0057] Then PCR amplification was carried out on the 7500Fast quantitative PCR instrument of American life technology company.

[0058] Reaction conditions: pre-denaturation at 95°C for 2min, denaturation at 95°C for 30S, annealing at 60°C for 30S, 40 cycles.

[0059...

Embodiment 3

[0060] Example 3: Specificity evaluation of live Cronobacter sakazakii real-time fluorescent PCR method in infant formula milk powder 1. Strain detection:

[0061] (1) Sample EMA pretreatment: use 1 standard strain of Cronobacter sakazakii, 15 isolates of Cronobacter sakazakii and 72 strains of non-Cronobacter sakazakii (Shigella, Vibrio parahaemolyticus) , Escherichia coli, Listeria monocytogenes, Salmonella, Staphylococcus aureus, etc.) bacteria suspension, adding the EMA in the kit provided in Example 1 to make the final concentration 50 μg / ml. After being placed in a dark place for 5 minutes, light treatment was carried out with a 650W halogen light source for 5 minutes. The light source was placed 20 cm away from the sample tube, and the EP tube was placed on ice to prevent the sample temperature from being too high. Under the same conditions, the dark place and the light treatment were repeated 3 times, and then precipitated with saline 3 times.

[0062] (2) Nucleic aci...

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Abstract

The invention relates to the field of food, in particular to a kit and an application thereof as well as a detection method. According to the invention, a method for detecting live cronobacter sakazakii in infant formula milk powder by using an EMA fluorescent quantitative PCR technology; a sample of infant formula milk powder polluted by cronobacter sakazakii is subjected to double-blind detection and simulation, and the tests for the removal rate of dead bacteria DNA, sensitivity and specificity indicate that the method is feasible. According to the method, EMA is used for the pretreatment of the detected sample, so as to avoid the false positive rate caused by dead bacteria or residual DNA in conventional PCR amplification and greatly improve the specificity. Moreover, the fluorescent PCR amplification technology of TaqMan-MGB probe is adopted, so as to greatly improve the bacterial sensitivity and specificity.

Description

technical field [0001] The invention relates to the field of food, in particular to a kit and its application and detection method. Background technique [0002] Cronobacter sakazakii is a motile, non-spore-forming, facultatively anaerobic, Gram-negative bacillus with periosteinous flagella that can cause infant mortality, especially in premature, low-birth-weight infants or those who are immune Damaged infants pose the greatest threat. Severe cases can lead to sepsis, meningitis, or necrotizing enterocolitis. The fatality rate in reported cases is 20% to 50%. Survivors often have severe neurological sequelae. Epidemiological studies have shown that contaminated infant formula is the main channel for infants and premature infants to be infected with Cronobacter sakazakii. [0003] At present, the detection method of Cronobacter sakazakii in infant formula milk powder is mainly separated and identified by culture method. The whole process is cumbersome, time-consuming, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
Inventor 周蒂范丽华梅玲玲
Owner 贝因美股份有限公司
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