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Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes

A technology for mononucleosis and staphylococcus, which is applied in the field of microbiological testing, can solve the problems of poor specificity, high laboratory safety requirements, and long time, and achieve the effect of simple method

Inactive Publication Date: 2010-11-10
中华人民共和国徐州出入境检验检疫局
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Problems solved by technology

(1) Bacterial culture, the results are highly reliable, and bacterial drug susceptibility tests can be done, but it takes too long and the application is limited. At the same time, due to Staphylococcus aureus, Salmonella, Shigella and Listeria Bacteria, etc. are very harmful, and have high requirements for laboratory safety, which is not conducive to the general screening of the above-mentioned bacteria.
(2) Polymerase chain reaction (PCR), which has poor specificity and has the advantage that the sensitivity can reach 98% to 100%. However, it requires PCR amplifiers, gel electrophoresis and other related expensive equipment, which is not conducive to primary medical epidemic prevention Institutional and on-site rapid determination

Method used

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  • Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes
  • Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes
  • Nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes

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Embodiment

[0040] (1) Nucleic acid extraction: absorb 1ml of the bacterial suspension cultured on a shaker at 37°C for 10 hours, centrifuge at 10,000rpm for 5min, discard the supernatant, add 100μl of sterile water and shake well (or store it in a refrigerator at 4°C) Pick 2-3 colonies on the plate and put them in 100 μl sterile water). Boil at 100°C for 10 minutes, centrifuge at 10,000 rpm for 5 minutes with a desktop centrifuge, and the supernatant is genomic DNA, which can be directly used as a template for nucleic acid amplification reaction.

[0041] (2) LAMP reaction: Prepare the above reaction system, mix the reaction system, and distribute it into 0.2ml PCR reaction tubes, 19ul per tube, add the template extracted in step (1) to each PCR reaction tube, 1ul per tube , 1ul each of the positive control substance and the negative control substance were used as the positive control and the negative control respectively, and mixed evenly. Put it in a constant temperature water bath, s...

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Abstract

The invention discloses a nucleic acid screening method of staphylococcus aureus, salmonella, shigella and listeria monocytogenes, belonging to the field of biological assay. In the method, by virtue of a loop-mediated isothermal amplification (LAMP) technology and a specific primer, the specific area of target genes is amplified by an LAMP technical platform; and with the aid of a positive and negative quality control system and an internal control detection system, nucleic acid screening or detection is carried out on the staphylococcus aureus, the salmonella, the shigella and the listeria monocytogenes from the molecular level. The nucleic acid screening method of the invention has the characteristics of being simple, convenient, economical, fast, sensitive and specific, and the method has wide application prospect.

Description

technical field [0001] The invention belongs to the field of microbial inspection, and in particular relates to a nucleic acid screening method for Staphylococcus aureus, Salmonella, Shigella and Listeria monocytogenes developed based on the principle of loop-mediated isothermal amplification (LAMP) technology . Background technique [0002] At present, the detection methods of Staphylococcus aureus, Salmonella, Shigella and Listeria monocytogenes are mainly microbial culture identification and multiplex PCR detection technology. (1) Bacterial culture, the results are highly reliable, and bacterial drug susceptibility tests can be done, but it takes too long and the application is limited. At the same time, due to Staphylococcus aureus, Salmonella, Shigella and Listeria Bacteria, etc. are very harmful and have high requirements for laboratory safety, which is not conducive to the general screening of the above-mentioned bacteria. (2) Polymerase chain reaction (PCR), which ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/445C12R1/42C12R1/01
Inventor 易海华邵景东宋阳威孙立新钱进房超吴萍兰杨志俊徐波孙慧宇王伟
Owner 中华人民共和国徐州出入境检验检疫局
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