阅读说明：本技术 一种结核免疫组化试剂盒在结核病病变组织诊断中的应用 (A kind of application of tuberculosis immunity group kit in the diagnosis of tuberculosis pathological tissues ) 是由 章晓联 于 2019-08-27 设计创作，主要内容包括：本发明公开了一种结核免疫组化试剂盒在结核病病变组织(包括肺内和肺外结核病变组织)诊断中的应用,属于结核病诊断领域。本发明采用一种结核抗原特异性适配体核酸“抗体”,采用生物素、或荧光、化学发光等标记,检测结核病变组织中存在的结核分枝杆菌特异抗原(ManLAM抗原),该抗原可存在于菌体及其分泌到周围空间；通过临床样本免疫组化证实,该结核抗原特异性适配体“抗体”在结核感染组织着色高,且背景着色低。所述结核病为结核分枝杆菌或耐药结核分枝杆菌导致的结核病(包括肺内和肺外结核病),以及所有人畜共患的结核病。本发明提供了一种结核免疫组化试剂盒在结核病病变组织,为结核病变组织的组织病理学确诊提供了新的工具。(The invention discloses a kind of application of tuberculosis immunity group kit in tuberculosis pathological tissues (becoming tissue including intrapulmonary and extrapulmonary tuberculosis) diagnosis, belong to diagnosis of tuberculosis field.The present invention uses a kind of tuberculosis antigen specificity aptamers nucleic acid " antibody ", it is marked using biotin or fluorescence, chemiluminescence etc., mycobacterium tuberculosis specific antigen (ManLAM antigen) present in tuberculosis tissue is detected, which may be present in thallus and its be secreted into surrounding space；It is confirmed by clinical sample immunohistochemistry, which colours height in tuberculosis infection tissue, and background coloration is low.The tuberculosis is tuberculosis (including intrapulmonary and extrapulmonary tuberculosis) and the tuberculosis of all infecting both domestic animals and humans caused by mycobacterium tuberculosis or Drug-Resistant Mycobacterium tuberculosis.The present invention provides a kind of tuberculosis immunity group kits in tuberculosis pathological tissues, makes a definite diagnosis for the histopathology of tuberculosis tissue and provides new tool.)

1. a kind of application of tuberculosis immunity group kit in tuberculosis lesion (intrapulmonary and the outer pathological tissues of lung) organizational diagnosis.

2. application according to claim 1, a kind of kit of the tissue section strain for mycobacterium tuberculosis detection, It is characterized by comprising aptamer (sons), and the label such as biotin, fluorescence or chemiluminescence dye can be used.

3. according to claim 1 with 2 aptamers " antibody ", it is characterised in that the aptamers " antibody " be with tuberculosis it is special Property glycolipid antigen forms for drone design, and nucleotide sequence is as shown in SEQ ID No.1.

4. application according to claim 1, a kind of kit of the tissue section strain for mycobacterium tuberculosis detection, It is characterized by comprising the labeling nucleic acids such as biotin, fluorescence or chemiluminescence dye aptamers (son), cleaning solution, substrate solutions Deng.

5. application described in -4 according to claim 1, immunohistochemical staining method, include the following steps:

1) by aptamers " antibody " solution reaction of histotomy to be detected and biotin labeling, anti-binding slice is obtained；

2) by anti-binding slice and secondary antibody (Streptavidin-HRP) solution reaction, two anti-bindings slice is obtained；

3) two anti-binding slice is dyed with DAB dyeing liquor, obtains DAB stained slice (immunohistochemical staining)；

4) [optional step] dyes the DAB stained slice with carbolic acid azaleine dyeing liquor, obtains the dyeing of carbolic acid azaleine and cuts Piece (acid-fast stain)；

5) the carbolic acid azaleine stained slice is dyed with haematoxylin dyeing liquid, obtains stained slice, realize histotomy dye Color (is redyed)；

The histotomy to be detected is the histotomies to be detected such as paraffin embedding or frozen section.

6. application described in -4 according to claim 1, fluorescent staining method detection method, include the following steps:

1) by aptamers " antibody " solution reaction of histotomy to be detected and fluorescent dye (such as AF488) label, fluorescence is obtained In conjunction with slice；

2) DAPI dyes nucleus (optional) is used；

3) with fluorescence microscope as a result, the positive is green fluorescence

The histotomy to be detected is the histotomies to be detected such as paraffin embedding or frozen section.

Technical field

The present invention relates to diagnosis of tuberculosis field, a kind of tuberculosis immunity group kit is provided in tuberculosis (including lung Interior and extrapulmonary tuberculosis) application in pathological tissues diagnosis is the tissue of tuberculosis and infecting both domestic animals and human tuberculosis pathological tissues Pathology, which are made a definite diagnosis, provides new tool.

Background technique

Tuberculosis is still the chronic infectious disease for seriously endangering human health at present, and tuberculosis caused by mycobacterium tuberculosis is people Raise the tuberculosis suffered from altogether.The whole world has the people of nearly one third to infect mycobacterium tuberculosis at present, according to WHO " 2018 global tuberculosis reports Accuse " 2017 Nian Xinfa tuberculosis cases 10,000,000 of estimation, because tuberculosis death toll is up to 1,300,000, more than other Death of Infectious Diseases The summation of number.

Early stage and specific diagnosis have extremely important meaning to tuberculosis therapy, prognosis and the control of propagation.Mesh The preceding diagnosis of tuberculosis technology clinically applied mainly have tuberculin (PPD) test, acid-fast stain, serology antibody test, , there is more or less limitation in the methods of the ELISpot detection of IFN-γ and genetic chip, e.g., false in clinical application Positive rate is high, cumbersome etc., and especially these methods cannot function as tuberculosis and make a definite diagnosis foundation；And can as tuberculosis make a definite diagnosis according to According to Sputum bacterial culture and PCR method: culture is same cumbersome, and takes long time, thus often can not guiding clinical treatment, It is relatively low additionally, due to the current China's tuberculosis culture positive rate of a variety of causes, influence the promptly and accurately diagnosis of tuberculosis；PCR method is opposite It is time-consuming shorter, but there are costly, and cannot distinguish between and previously be cured infection and newly sent out active tuberculosis.Therefore, tuberculosis is improved Diagnosis rate is significant.It is marked according to " People's Republic of China's health industry standard-diagnosis of pulmonary tuberculosis " (WS288-2017) Standard, the lung tissue immunopathology inspection positive also can be used as tuberculosis and make a definite diagnosis foundation.Therefore exploitation tuberculosis pathological tissues tissue Pathology detection method has important benefit for improving tuberculosis diagnosis rate.

Lipolysaccharide is the main component of mycobacterium tuberculosis cell wall, and about 30% M. tuberculosis genes take part in lipid Synthesis and metabolism.Mannose-modified fat arabian mannan (Mannosylated lipoarabinomannan, ManLAM the lipolysaccharide) being primarily present in pathogenic bacillus tuberculosis typus humanus's cell wall.Research shows that mycobacterium tuberculosis living is not Disconnected release ManLAM, therefore ManLAM is not only expressed in thallus, is also expressed in its surrounding space, distribution space is wide, is consequently adapted to Target is detected as tuberculosis tissue (including intrapulmonary and extrapulmonary tuberculosis become tissue) immunohistochemistry, it is strong color signal to be improved Degree and range.It there is no the ImmunohistochemistryMethods Methods report of targeting tulase surface glycolipid at present.

Summary of the invention

Based on this, the purpose of the present invention is a kind of tuberculosis immunity group kits (including to tie outside intrapulmonary and lung in tuberculosis Core disease) pathological tissues diagnosis in application, be tuberculosis (including tuberculosis outside intrapulmonary and lung) pathological tissues histopathology it is true It examines and new tool is provided.

Specific technical solution is as follows:

It is an object of the present invention to provide it is a kind of for mycobacterium tuberculosis detection tissue section strain kit, Including DAB dyeing liquor, carbolic acid azaleine dyeing liquor, haematoxylin dyeing liquid, aptamers " antibody ".

The present invention provides the tissue section strain method of mycobacterium tuberculosis detection, includes the following steps:

1) by histotomy to be detected and aptamers " antibody " solution reaction, anti-binding slice is obtained；

2) by anti-binding slice and secondary antibody (Streptavidin-HRP) solution reaction, two anti-bindings is obtained and are cut Piece；

3) two anti-binding slice is dyed with DAB dyeing liquor, obtains DAB stained slice (immunohistochemistry dye Color)；

4) [optional step] dyes the DAB stained slice with carbolic acid azaleine dyeing liquor, obtains carbolic acid azaleine dye Color is sliced (acid-fast stain)；

5) the carbolic acid azaleine stained slice is dyed with haematoxylin dyeing liquid, obtains stained slice, realize that tissue is cut Piece dyes (redying)；

The aptamers " antibody " are to form using tuberculosis specific glycolipids antigen as drone design.

It is a further object to provide the kits for tuberculosis tissue (including intrapulmonary and extrapulmonary tuberculosis Become tissue) immunodiagnosis application.

Beneficial effects of the present invention:

There are various defects for the means of tuberculosis laboratory diagnosis at present, cause tuberculosis diagnosis rate low.Most of tuberculosis are examined It is disconnected only to carry out clinical diagnosis in conjunction with testing results such as iconography detection, T-SPOT only in accordance with clinical symptoms, according to " the Chinese people Republic's health industry standard-diagnosis of pulmonary tuberculosis " (WS288-2017) standard, it is made a definite diagnosis, be can be improved using Histopathological method Tuberculosis diagnosis rate.A kind of tuberculosis antigen specificity tuberculosis immunity group kit disclosed by the invention can be effective for tuberculosis Become the histopathological diagnosis of tissue (becoming tissue including intrapulmonary and extrapulmonary tuberculosis), tuberculosis antigen detected is expressed in thallus And its surrounding space, distribution space is wide, improves color signal intensity and range；It is confirmed by clinical sample immunohistochemistry, it should Tuberculosis antigen specificity aptamers " antibody " colour height in tuberculosis infection tissue, and background coloration is low.It can be improved lungy Diagnosis rate is diagnosed, and the validity of tuberculosis prophylaxis and control can be improved, to be laid for preferably prevention and control tuberculosis Basis.

Detailed description of the invention

Fig. 1 intestinal tuberculosis pathological tissues slice immunohistochemical staining+acid-fast stain+is redyed and acid-fast stain+redye two methods Compare.It is positive region that black arrow, which show acid-fast stain, and the visible acid-fast stain of right figure is Chong Die with immunohistochemical staining.

Fig. 2 aptamers " antibody " immunohistochemistry detection effect is verified compared with other antibody mediated immunity group detection effects.

Fig. 3 aptamers " antibody " ImmunohistochemistryMethods Methods are detected for clinical pathology histotomy.

Specific embodiment

Illustrate the present invention below in conjunction with the drawings and specific embodiments.

Unless otherwise specified, actually derived from used in following embodiment it is commercially available, operating method be it is existing often Advise operating method.