Immuno-fluorescent staining method for detecting mycobacterium tuberculosis in leukocytes and kit

Abstract

The invention discloses a method for rapidly detecting mycobacterium tuberculosis in blood (blood plasma and blood cells) through fluorescent staining and a kit.ESAT-6 and CFP-10 antigens of the mycobacterium tuberculosis in the blood are specifically detected by using monoclonal antibodies of RD-1 zone ESAT-6 and CFP-10 antigens of the mycobacterium and are used for etiologic detection and early-stage and rapid clinical diagnosis of tuberculosis diseases.According to the method, the blood is divided into the blood plasma and the blood cells, the mycobacterium tuberculosis enriched in the blood plasma is separated by using a micro-fluid device based on a membrane, meanwhile a slide is coated with hemocytes (leukocytes), and the mycobacterium tuberculosis in the blood plasma and the leukocytes are detected by using the monoclonal antibodies of the ESAT-6 and CFP-10 antigens and adopting direct and indirect fluorescent staining methods.The method is simple in operation, high in sensitivity and strong in specificity and is a definite tuberculosis etiology diagnosis method, and the kit for detecting the mycobacterium tuberculosis in the blood is developed.In addition, by means of the method, pathogenic mycobacterium tuberculosis infection can be detected, and theoretically non-pathogenic mycobacterium tuberculosis produced in blood due to bacillus calmette guerin vaccine inoculation can be also distinguished.

Description

technical field [0001] An immunofluorescent staining method and kit for detecting Mycobacterium tuberculosis in white blood cells. Background technique [0002] Tuberculosis is a chronic infectious disease caused by a single pathogen, Mycobacterium tuberculosis. In recent years, due to factors such as the increase of population mobility, the emergence of drug-resistant tuberculosis, and the double infection of tuberculosis and HIV, tuberculosis has made a comeback and has once again become a major public health and social problem that seriously endangers the health of the people. According to the report of the World Health Organization (WHO), it is currently estimated that about 2 billion people in the world are infected with Mycobacterium tuberculosis, with 9 million new cases in 2013. my country is a country with a high burden of tuberculosis. The number of tuberculosis patients ranks second in the world, with an annual incidence of about 1.3 million, accounting for 14% o...

AI Technical Summary

Problems solved by technology

[0010] First, the detection is based on the patient's sputum sample. Factors such as the presence or absence of the patient's sputum, whether the collection method is correct, whether the quality is good or bad, and whether it is sent for inspection in a timely manner will directly affect the test results, resulting in the inability to obtain bacteriological tests based on sputum samples. With higher detection rate and better specificity, new detection methods are needed to replace traditional sputum detection

[0011] Second, pulmonary tuberculosis patients account for 80% of all tuberculosis patients, and nearly 50% of them are patients with negative sputum bacteria. The traditional bacteriological detection of sputum bacteria will inevitably lead to missed diagnosis of this part of the population, and other methods are needed to assist diagnosis; the other 20% Most tuberculosis patients present with extrapulmonary tuberculosis, and sputum samples cannot be obtained, so no clear bacteriological evidence can be obtained. They can only rely on other methods for auxiliary diagnosis, or tentative anti-tuberculosis treatment. So far, new drugs suitable for bacterial negative and extrapulmonary tuberculosis are needed. Bacteriological detection method

However, the above methods all use DNA as a template, and the stability of DNA makes it impossible to distinguish live bacteria from dead bacteria in the PCR test results, resulting in false positive results.

[0025] However, compared with sputum smear testing, molecular testing is still more complicated, and it is also mainly based on sputum samples. At present, there is no relevant research on the detection results of molecular diagnostic methods for blood samples.

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