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Fluorescence in situ hybridization detection kit for detection of Mycobacterium tuberculosis infection

A fluorescent in situ hybridization and detection kit technology, which is applied in the field of medical detection, can solve the problems of poor specificity, false negatives, insufficient diagnostic techniques, etc., and achieve the effect of detection sensitivity

Active Publication Date: 2016-08-24
广州华银医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Histology has atypical lesion morphology, which is similar to sarcoidosis, lung cancer, pneumonia, etc., and some bacterial infections can also cause tissue morphology similar to tuberculosis, so it is easy to misdiagnose
Because acid-fast staining is aimed at all acid-fast bacilli, the specificity is not strong, and those who stain positive must be identified from other non-pathogenic acid-fast bacilli, and are greatly affected by experimental conditions, such as dewaxing, alcohol, etc. , initial staining temperature, background, etc., it is easy to have false negative results, resulting in misdiagnosis
For a long time, due to the lack of diagnostic technology, about 50% of tuberculosis patients in the country have not been diagnosed and treated in time, posing a huge threat to healthy people

Method used

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  • Fluorescence in situ hybridization detection kit for detection of Mycobacterium tuberculosis infection
  • Fluorescence in situ hybridization detection kit for detection of Mycobacterium tuberculosis infection

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Experimental program
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Effect test

Embodiment 1

[0028] The preparation of embodiment 1 specificity probe

[0029] According to the human tuberculosis gene sequence reported by NCBI, homology comparison analysis was carried out to find the most conserved region of the gene, and IS6110, MPRT40 and 16s rRNA genes were selected as target sequences, and primer5.0, oligo6.0 and Primer Express2.0 software, designed three groups of fluorescent probes specific to Bacillus. The probe was put into NCBI for BLAST homology analysis, and the result showed that it was heterologous to other gene sequences, thereby avoiding the existence of non-specificity during hybridization. The probe was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0030] The sequences of the three oligonucleotide probes are respectively shown in SEQ ID NO: 1-3.

[0031] Preparation of fluorescently labeled probes: FITC fluorescein was selected, and the probes were directly labeled by the gap translation method. Directly attaching fluorescein to the pr...

Embodiment 2

[0041] The preparation of embodiment 2 detection kits

[0042] Hybridization solution composition: 10% (w / v) dextran sulfate, 10mM NaCl, 20% (v / v) formamide, 0.1% (w / v) sodium pyrophosphate, 0.2% (w / v) polyvinyl Pyrrolidone, 5mM Na2EDTA, 0.1% (v / v) TritonX-100, 50mM Tris / HCl (pH7.5); 30pM fluorescently labeled three specific probes (prepared in Example 1).

[0043] Preparation of DAPI counterstain: Take 2.2 mg of DAPI powder, add 550 μl of distilled water and mix well to make a DAPI stock solution of 4 mg / ml. Store at 4°C. Working solution: Take 2.5μl DAPI stock solution and add it to 5ml distilled water, mix well, 2μg / ml.

Embodiment 3

[0044] The application of embodiment 3 detection kits

[0045] Sputum samples and lesion tissue samples were taken from the same patient, a total of 20 cases.

[0046]The sputum samples were cultured by the Roche culture method, specifically, the modified Lowenstein-Jensen medium was selected to culture the treated sputum. Specifically, add 2 mL of 4% sodium hydroxide to 1 mL of sputum sample, vortex and oscillate for 30 seconds to mix, and let stand at room temperature for 20 minutes, during which time oscillate 2 to 3 times to promote liquefaction of sputum. Take 0.1mL of digested sputum with a sterile graduated capillary pipette, inoculate slowly and evenly on the slant of each culture medium, and culture in a 37°C incubator. Observe the culture situation on the 3rd and 7th day after inoculation, and then observe once a week until the end of the 8th day. If there is a positive culture, report it at any time. If no bacterial growth is found after 8 weeks of culture, the re...

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Abstract

The invention relates to a fluorescence in situ hybridization detection kit for detecting mycobacterium tuberculosis infection in a phthisis pathological tissue slice. The fluorescence in situ hybridization detection kit for detecting mycobacterium tuberculosis infection comprises a hybridization solution, a denaturation solution, a DAPI afterstain, and a packaging box packing reagent bottles or reagent tubes in an isolating and concentrating manner. The hybridization solution of the fluorescence in situ hybridization detection kit contains three special probes, namely, IS6110 probe with the sequence shown in SEQ ID NO:1, MPRT40 probe with the sequence shown in SEQ ID NO:2, and 16s rRNA probe with the sequence shown in SEQ ID NO:3.

Description

technical field [0001] The invention belongs to the field of medical detection, in particular to a fluorescent in situ hybridization detection kit for detection of Mycobacterium tuberculosis infection. Background technique [0002] Tuberculosis is listed as one of the major infectious diseases in my country and is a respiratory infectious disease that seriously endangers the health of the people. According to the results of the Fifth National Tuberculosis Epidemiological Sampling Survey conducted by the Ministry of Health, the annual number of tuberculosis cases in my country is about 1.3 million, accounting for 14.3% of global cases and ranking second in the world. From 2001 to 2010, a total of 8.28 million cases of tuberculosis were detected and treated nationwide, including 4.5 million cases of infectious tuberculosis. [0003] Traditionally, there are three ways to diagnose pulmonary tuberculosis: (1) Mycobacterium tuberculosis is detected in sputum and diseased tissues...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/32
CPCC12Q1/6841C12Q1/6895C12Q2543/10
Inventor 温韵洁王晓丹孙宇超徐蕙
Owner 广州华银医学检验中心有限公司
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