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Special bacterial genome DNA extraction kit and method for septicemia

A kit and genome technology, applied in the fields of molecular diagnosis and molecular biology, can solve the problems of operator poisoning, difficulty in effectively eliminating human genome DNA interference, etc., and achieve high-quality results

Inactive Publication Date: 2018-03-02
南通柯侎克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When this method extracts DNA from septic blood, it is difficult to effectively eliminate the interference of human genomic DNA, and the organic reagents such as phenol and chloroform used have a certain toxic effect on the operator.

Method used

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  • Special bacterial genome DNA extraction kit and method for septicemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Using the extraction of bacterial genome DNA in normal human blood, the method is as follows:

[0027] A. Select 1-5ml of fresh blood, add 0.3 times the volume of buffer GRA, let it stand at room temperature for 10 minutes, centrifuge at 2000rpm for 5 minutes, and discard the upper layer;

[0028] B. Repeat operation step A three times;

[0029] C. Add an equal volume of buffer solution GRB to the lower layer liquid, add it to the centrifuge and mix well; centrifuge at 12000rpm for 2min; discard the supernatant;

[0030] D. Repeat step C for a total of 2 to 3 times until the resulting precipitate has no red color;

[0031] E. Add 200ul bacterial lysate GA to the precipitate, shake and suspend;

[0032] F. Add 20ul proteinase K solution to the above liquid, mix well, and bathe in water at 65°C for 0.5-3h;

[0033] G. Add 200 μl bacterial lysate DB, mix well, and bathe in water at 70°C for 10 minutes, and the solution becomes clear at this time;

[0034] H. Add 200 μl...

Embodiment 2

[0042] Add 10,000 Staphylococcus aureus to each ml of blood to extract bacterial genomic DNA from normal human blood, as follows:

[0043] A. Select 1-5ml of fresh blood, add 0.3 times the volume of buffer GRA, let it stand at room temperature for 10 minutes, centrifuge at 2000rpm for 5 minutes, and discard the upper layer;

[0044] B. Repeat operation step A three times;

[0045] C. Add an equal volume of buffer solution GRB to the lower layer liquid, add it to the centrifuge and mix well; centrifuge at 12000rpm for 2min; discard the supernatant;

[0046] D. Repeat step C for a total of 2 to 3 times until the resulting precipitate has no red color;

[0047] E. Add 200ul bacterial lysate GA to the precipitate, shake and suspend;

[0048] F. Add 20ul proteinase K solution to the above liquid, mix well, and bathe in water at 65°C for 0.5-3h;

[0049] G. Add 200 μl bacterial lysate DB, mix well, and bathe in water at 70°C for 10 minutes, and the solution becomes clear at this ...

Embodiment 3

[0058] Normal human blood is added to the extraction of bacterial genomic DNA in Escherichia coli, and 10,000 Staphylococcus aureus are added to each ml of blood, the method is as follows:

[0059] A. Select 1-5ml of fresh blood, add 0.3 times the volume of buffer GRA, let it stand at room temperature for 10 minutes, centrifuge at 2000rpm for 5 minutes, and discard the upper layer;

[0060] B. Repeat operation step A three times;

[0061] C. Add an equal volume of buffer solution GRB to the lower layer liquid, add it to the centrifuge and mix well; centrifuge at 12000rpm for 2min; discard the supernatant;

[0062] D. Repeat step C for a total of 2 to 3 times until the resulting precipitate has no red color;

[0063] E. Add 200ul bacterial lysate GA to the precipitate, shake and suspend;

[0064] F. Add 20ul proteinase K solution to the above liquid, mix well, and bathe in water at 65°C for 0.5-3h;

[0065] G. Add 200 μl bacterial lysate DB, mix well, and bathe in water at 7...

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Abstract

The invention discloses a special bacterial genome DNA extraction kit and method for septicemia. The kit consists of a buffer solution GPA, a buffer solution GRB, a bacterial lysis solution DA, a bacterial lysis solution DB, a buffer solution DC, a washing solution PE, an eluent EB and protease K. According to the extraction kit and method provided by the invention, red / white cells are separated by virtue of the buffer solution GRB, so that the influence and interference of human genome can be removed to the greatest extent; bacterial cells are cracked and broken by virtue of SDS and the protease K, and protein is degraded, so that bacterial genome DNA is released; and by virtue of a silica gel column separation and purification method, the bacterial genome DNA with high quality can be obtained.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and molecular diagnosis, in particular to a bacterial genome DNA extraction kit and method specially used for sepsis. Background technique [0002] Sepsis is when pathogenic bacteria invade the blood system and can grow and reproduce in the blood, producing a large amount of toxins and causing acute systemic infection. The disease is very harmful and can easily lead to death. Therefore, the rapid determination of the type of bacteria that causes sepsis can guide the selection and use of antibiotics, reduce blind drug use, and shorten the treatment time of patients. The general procedure for the extraction and purification of bacterial genomic DNA from septic blood is: isolation of red / white blood cells, red blood cell lysis, bacterial lysis, and purification of bacterial genomic DNA. The classic bacterial DNA extraction method is phenol-chloroform extraction. When this method extract...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 杨天逸
Owner 南通柯侎克生物科技有限公司
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