Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Fluorescence in situ hybridization detection kit for detecting mycobacterium tuberculosis infection

A fluorescence in situ hybridization and detection kit technology, applied in the field of medical detection, can solve the problems of weak specificity, atypical lesions, false negatives, etc., and achieve the effect of sensitive detection

Active Publication Date: 2015-11-18
广州华银医学检验中心有限公司
View PDF3 Cites 1 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fluorescence in situ hybridization detection kit for detecting mycobacterium tuberculosis infection
  • Fluorescence in situ hybridization detection kit for detecting mycobacterium tuberculosis infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation of specific probe

[0029] According to the human Mycobacterium tuberculosis gene sequence reported by NCBI, homology comparison analysis was performed to find the most conserved region of the gene. IS6110, MPRT40 and 16srRNA genes were selected as target sequences, and primer5.0, oligo6.0 and Primerexpress2 were used in combination. 0 software, designed three sets of binding bacillus-specific fluorescent probes. Put the probe into NCBI for BLAST homology analysis, the results showed that it was heterologous to other gene sequences, thus avoiding the non-specific existence during hybridization. The probe was synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.

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

[0031] Preparation of fluorescently labeled probe: FITC fluorescein is selected, and the probe is directly labeled by the notch translation method. Fluorescein directly with the probe ...

Embodiment 2

[0041] Example 2 Preparation of detection kit

[0042] Hybrid solution composition: 10% (w / v) dextran sulfate, 10 mM NaCl, 20% (v / v) formamide, 0.1% (w / v) sodium pyrophosphate, 0.2% (w / v) polyvinyl pyrrolidone , 5mMNa2EDTA, 0.1%(v / v) TritonX-100, 50mMTris / HCl (pH 7.5); three specific probes (prepared in Example 1) each with 30pM fluorescently labeled.

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

Embodiment 3

[0044] Example 3 Application of detection kit

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

[0046] The sputum sample adopts the Roche culture method, specifically, the modified Lowenstein-Jensen medium is used to culture the processed sputum. Specifically, 1 mL of a sputum specimen was added to 2 mL of 4% sodium hydroxide, vortexed for 30 seconds to mix, and allowed to stand at room temperature for 20 minutes, during which it was shaken 2 to 3 times to promote liquefaction of the sputum. Use a sterile graduated capillary pipette to take 0.1 mL of digested sputum, slowly and evenly inoculate each medium slant, and place it in a 37°C incubator for culture. Observe the culture on the 3rd and 7th day after inoculation, and then observe once a week until the 8th weekend. If the culture is positive, report it at any time. No bacterial growth is seen until 8 weeks after culture, and the report is culture negative.

[0047] The...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com