Rapid purification device and rapid purification method for pathogenic bacteria in sputum sample

A pathogenic and rapid technology, applied in the field of medical diagnosis, can solve the problems of low separation efficiency and cannot meet the purification of pathogenic bacteria, etc., and achieves the effects of fast flow rate, convenient operation and simple preparation.

Active Publication Date: 2017-08-25
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, these chips still have disadvantages such as the need for multi-channel fluid con

Method used

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  • Rapid purification device and rapid purification method for pathogenic bacteria in sputum sample
  • Rapid purification device and rapid purification method for pathogenic bacteria in sputum sample
  • Rapid purification device and rapid purification method for pathogenic bacteria in sputum sample

Examples

Experimental program
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Example Embodiment

[0038] Example 1 Chip structure and preparation

[0039] see figure 1 , the rapid purification device includes a microfluidic chip with an upper, middle and lower three-layer structure. The middle-layer chip 2 is provided with a main channel 203, a waste liquid collection channel 202 and a pathogenic bacteria collection channel 201; in this embodiment, a string structure composed of 8 series units and 12 pathogenic bacteria collection channels are arranged on the middle-layer chip 2. The like structure and the pathogen collection channel are arranged in parallel. One end of the upper chip 1 is provided with a liquid inlet hole 101, and the other end is provided with a pathogenic bacteria collection hole 102 and a waste liquid collection hole 103. The liquid inlet hole communicates with one end of the string structure of the separation unit, and the pathogenic bacteria collection hole In communication with the pathogenic bacteria collection channel, the waste liquid collectio...

Example Embodiment

[0047] Example 2: Use of chips and detection of separation efficiency

[0048] KYSE30-GFP cells and red light-labeled bacteria T23101-cherry were used to simulate exfoliated cells and pathogenic bacteria in sputum, which were mixed in a certain quantity ratio, and the separation efficiency of cells and bacteria was detected by rapid purification device chip separation. The specific steps are: trypsin digestion and stable transfection of GFP esophageal cancer cells KYSE30-GFP (green light), add 10% FBS RPM1640 medium to stop, centrifuge at 1000 rpm for 5 min, remove the supernatant, add PBS containing 4% fetal bovine serum (PH= 7.4), cell count, adjust cell concentration 0.5-1X10 6 pcs / ml. Pick the red-marked bacteria T23101-cherry into 2 ml of LB medium (AMP+) and culture at 37°C at 200 rpm overnight, centrifuge at 4000 rpm for 5 min, remove the supernatant, add PBS to vortex to suspend the bacteria, dilute the bacterial solution twice, and count the bacteria.

[0049] Take ...

Example Embodiment

[0053] Example 3: Detection of separation efficiency of sputum samples

[0054] After the sputum samples were liquefied by the Saccomanno-DTT method, the bacteria BW25113 (GFP+) was added and then passed into the device for rapid purification of pathogenic bacteria in the sputum samples for separation. The operation method is the same as that of Example 2. After the liquefied sputum was separated, Hoechst 33342 (100×) was added for staining for 10 min, centrifuged at 3000 rpm for 3 min, washed once with PBS, suspended in 1 / 50 volume of PBS, and 1 μl was dropped on a glass slide. The performance of the analysis chip to separate cells and bacteria from liquefied sputum, see Image 6 . At the same time, the GFP gene was used to represent bacteria, and the human BTF gene was used to represent cells. RT-PCR was performed on the original and purified collection samples to detect and analyze the separation efficiency of cells and bacteria in sputum samples.

[0055] GFP primer (PC...

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Abstract

The invention provides a rapid purification device for pathogenic bacteria in a sputum sample. The rapid purification device comprises a pathogenic bacterium collecting channel and a main channel composed of separation units which are connected in series, wherein multiple separation units connected in series form a string-shaped structure, and the string-shaped structure is parallel to the pathogenic bacterium collecting channel; each separation unit comprises an arc-shaped part with at least partial projection toward the pathogenic bacterium collecting channel, and the projection of the arc-shaped part is connected with the pathogenic bacterium collecting channel by virtue of a fence. The invention also provides a rapid purification method for the pathogenic bacteria in the sputum sample. The provided rapid purification device for the pathogenic bacteria in the sputum sample is easy to operate, and a sample only needs to be simply pushed over a chip during operation. The stability is high, the efficiency for removing animal cells through separation is stabilized to be more than 99% for samples with different flow rates, different viscosities and different bacterium rates, and the collected pathogenic bacteria can keep the level close to half of the original sample.

Description

technical field [0001] The invention belongs to the field of medical diagnosis, and in particular relates to a separation device and a separation method for bacteria in a cell-containing sample. Background technique [0002] Human sputum samples are mainly composed of human secretions, human immune cells, pathogenic bacteria and other microorganisms, and are potentially an important source of information for non-invasive medical diagnosis. Before the use of antibiotics, it is often necessary to culture and analyze the resistance of pathogenic bacteria to avoid the abuse of antibiotics. In this process, the cultivation of pathogenic bacteria is difficult, and it takes a lot of time before treatment. At present, in the process of genome analysis of sputum pathogenic bacteria, or real-time quantitative PCR detection using microdroplet technology, there is a problem that it is difficult for samples to remove a large number of human immune cells in sputum, and it is difficult fo...

Claims

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Application Information

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IPC IPC(8): C12M1/00C12N1/20C12N1/02
CPCC12M23/16C12M45/04C12N1/02C12N1/20
Inventor 康禹罗春雄邵长君吴天于军林强王建楚亚男付荣荣
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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