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LAMP (loop-mediated isothermal amplification) rapid detection kit and detection method for salmonella

A technology for detection kits and Salmonella, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, and resistance to vector-borne diseases, etc. Insufficient system stability and other problems, to achieve the effect of convenient on-site application, high accuracy and good sensitivity

Inactive Publication Date: 2011-09-28
镇江出入境检验检疫局检验检疫综合技术中心
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AI Technical Summary

Problems solved by technology

[0006] There are already Salmonella LAMP detection kits and detection methods in the prior art. In the actual use process, there are still defects such as unreasonable configuration of the detection kit, insufficient stability of the reaction system, and insufficient pertinence and sensitivity of the designed primers.

Method used

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Embodiment

[0037] Make the loop-mediated isothermal amplification detection kit of Salmonella according to the following formula;

[0038] 1. Extract the Salmonella genome:

[0039] 1) Take 1mL Salmonella, 10000rpm, 5min, discard the supernatant.

[0040] 2) Precipitate bacteria, suspend in 600 μL TE, add 60 μL 10% SDS, 6 μL protein K10 mg / ml, mix well, 37 ° C, 1 h.

[0041] 3) Add 180 μL 5mol / L NaCl, mix well, then add 180 μL CTAB / NaCl solution, mix well, 65°C, 20min.

[0042] 4) Extract with an equal volume of phenol:chloroform (25:24), 12000rpm, 5min, transfer the supernatant to a clean EP tube.

[0043] 5) Repeat step 4.

[0044] 6) Take supernatant, about 500 μL, add 6 μL RNAse, 37°C, 30min.

[0045] 7) Add 1mL 100% absolute ethanol, 50μL 3M NaAc, -20°C, 90min.

[0046] 8), 12000rpm, 5min, discard the supernatant, add 200μL of 75% ethanol to wash the precipitate.

[0047] 9) Discard the supernatant, add 40 μL TE to dissolve the DNA, a white precipitate dissolves, and use it as...

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Abstract

The invention belongs to the field of biological products, and relates to a detection kit and a detection method for rapidly detecting salmonella by utilizing an LAMP (loop-mediated isothermal amplification) technique. The kit is composed of a salmonella genome extracting reagent and an LAMP reaction reagent, wherein the salmonella genome extracting reagent comprises the following components: SDS (sodium dodecyl sulfate), protease K, CTAB (cetyltrimethyl ammonium bromide) / NaCl solution and NaAC; and the LAMP reaction reagent is composed of a reaction liquid A and a reaction liquid B, the reaction liquid A comprises the following components: 10*Lampbuffer, Bst DNA (deoxyribonucleic acid) polymerase, dNTP (deoxy-ribonucleoside triphosphate), an interprimer 1, an interprimer 2, an outer primer 1, an outer primer 2, lycine and ultrapure water, and the reaction liquid B is a fluorescent dye. The invention can be used to carry out LAMP detection on salmonella, has the characteristics of rapid detection, high accuracy, high sensitivity and convenient field application, and can be widely applied in the fields of veterinarian, food and exit-entry quarantine, etc.

Description

technical field [0001] The invention belongs to the field of biological products, and relates to a detection kit and a detection method for rapidly detecting Salmonella by using a loop-mediated isothermal amplification (LAMP) technology. Background technique [0002] Salmonellosis is one of the zoonotic diseases of great significance in public health. According to the report of the World Health Organization, since 1985, the number of confirmed human illnesses caused by Salmonella has increased significantly worldwide. In some European countries have increased more than fivefold. In my country's inland areas, food poisoning caused by Salmonella has repeatedly ranked first. According to statistics, in my country's bacterial food poisoning, 70% to 80% are caused by Salmonella, and among the foods that cause Salmonella poisoning, more than 90% are animal products such as meat. Animal products contain a variety of rich nutrients, which are very suitable for the growth and repro...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10
CPCY02A50/30
Inventor 孙园园赵鹏吴小华朱金连刘骏王富海钱科
Owner 镇江出入境检验检疫局检验检疫综合技术中心
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