Method for detecting bacillus tubercle by adopting double-color flow cytometry

A technology of Mycobacterium tuberculosis and cytometry, which is applied in the detection field of Mycobacterium tuberculosis, can solve the problems of increased background noise, increased false positives, and inferior detection results, etc., to reduce high background noise, simplify detection steps, and reduce bacterial counts. Effects of Reunion and Loss

Inactive Publication Date: 2010-01-27
HUNAN UNIV
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Problems solved by technology

A research team once tried to use quantum dot-labeled antibodies combined with flow cytometry for the detection of Cryptosporidium eggs, but unexpectedly, the detection results were not as good as traditional organic dyes, mainly because quantum dots themselves brought a lot of False positive signal: On the one hand, the agglomeration of quantum dots significantly increases the background noise, which leads to an increase in false positives; on the other hand, the non-specific adsorption of quantum dots to particulate impurities in the sample causes increased false positives

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  • Method for detecting bacillus tubercle by adopting double-color flow cytometry
  • Method for detecting bacillus tubercle by adopting double-color flow cytometry
  • Method for detecting bacillus tubercle by adopting double-color flow cytometry

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Embodiment 1

[0029]Mix fluorescent nanoparticle-labeled Mycobacterium tuberculosis antibody with 6.0×10 6 The buffer solution of cells / mL Mycobacterium tuberculosis (this buffer solution is the pH 7.4, 0.01mol / L phosphate buffer solution containing the mass fraction of 1% bovine serum albumin and 0.05% Tween 80) was incubated at 37°C for 1h, The commercial stock solution of nucleic acid dye SYBR Green I was added to the solution after incubation at a final concentration of 10,000 times, and stained at room temperature for 15 minutes. The stained samples were directly analyzed by flow cytometry. Excited with the 488nm argon ion laser configured by the flow cytometer, use the FL1 detector (530+15nm bandpass filter) to detect the green fluorescence of the bacteria stained by SYBR Green I, use the FL3 detector (610nm long pass filter) filter) to detect the red fluorescence of fluorescent nanoparticles, and the analysis results are as follows figure 1 (F), shown by figure 1 (F) It can be seen...

Embodiment 2

[0039] Two-color flow cytometry using fluorescent nanoparticles and nucleic acid dye SYBR Green I was used to quickly detect Mycobacterium tuberculosis in synthetic urine samples. The detection steps and results are as follows:

[0040] Collect 23 midsection urines from different healthy people, take 1mL urine samples for each, and mix different concentrations of Mycobacterium tuberculosis into 18 of them respectively to make 6 groups containing 1.0×l0 4 cells / mL, 2.0×10 4 cells / mL, 3.0×10 4 cells / mL, 5.0×10 4 cells / mL, 1.0×10 5 cells / mL, 1.0×10 6 cells / mL synthetic urine samples of Mycobacterium tuberculosis, each group of synthetic urine samples contains three synthetic urine samples with the same concentration of Mycobacterium tuberculosis, and the remaining 5 urine samples are not mixed with Mycobacterium tuberculosis. Centrifuge each of the above samples at a speed of 12,000rpm for 5min, discard the supernatant, add 1mL of phosphate buffer, centrifuge and wash once ac...

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Abstract

The invention discloses a method for adopting the two-color flow cytometry to detect tubercle bacillus. The method is characterized in that: firstly, immunity co-breeding is performed on tubercle bacillus antibodies which are marked by fluorescent nano particles and a sample to be detected; secondly, the sample to be detected is dyed by nucleic aid dye; thirdly, the multiparameter flow cytometry is used for determining and analyzing the sample to be detected and analyzing and judging the detection result of the tubercle bacillus in the sample to be detected according to the number of objects provided with florescent double-positive signals which are detected in per unit volume. The method organically combines the signal amplifying function of the fluorescent nano particles and the flow cytometry which is capable of simultaneously performing quick detection with multipparameter, has the advantages of quickness and high sensitivity, and can greatly reduce the false positive rate of the detection through increase of dyeing of the nucleic acid dye.

Description

technical field [0001] The invention relates to a detection method for bacteria, in particular to a detection method for Mycobacterium tuberculosis. Background technique [0002] Tuberculosis caused by Mycobacterium tuberculosis infection is one of the important infectious diseases threatening human health. Since the mid-1980s, there has been an upward trend in the world. At present, one-third of the world's population has been infected by Mycobacterium tuberculosis, with more than 8 million new cases and more than 2 million deaths each year. Therefore, rapid and sensitive detection of Mycobacterium tuberculosis is of great significance for the diagnosis, management and control of tuberculosis. [0003] The traditional detection methods of Mycobacterium tuberculosis mainly include smear acid-fast staining method and culture method. At present, the diagnosis of tuberculosis in hospitals still relies on these two methods. The acid-fast staining method of smears is quick and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78C12Q1/68C12Q1/10
Inventor 王柯敏秦迪岚何晓晓谭蔚泓
Owner HUNAN UNIV
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