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A rapid detection kit and detection method for mycobacterium tuberculosis nasba-elisa in deer

A technology of Mycobacterium tuberculosis and detection kits, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of cumbersome and time-consuming separation, high detection cost of detection equipment, and limited diagnostic value, etc.

Active Publication Date: 2021-04-30
JILIN AGRI SCI & TECH COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the shortcomings of false positives and tedious and time-consuming pathogen isolation limit the diagnostic value of this method.
Nucleic acid methods, such as polymerase chain reaction (PCR), biological probes and gene chips, etc., are attracting increasing attention due to their sensitivity, accuracy and speed of detection, but they all require corresponding detection equipment and the detection costs are high and have not been widely promoted.

Method used

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  • A rapid detection kit and detection method for mycobacterium tuberculosis nasba-elisa in deer
  • A rapid detection kit and detection method for mycobacterium tuberculosis nasba-elisa in deer
  • A rapid detection kit and detection method for mycobacterium tuberculosis nasba-elisa in deer

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0078] Example 1: Mycobacterium tuberculosis NASBA kit specific detection:

[0079] Extract TB, HCV (hepatitis C virus), HIV-1 (human immunodeficiency virus-1), HBV (hepatitis B virus) RNA respectively, take 0.5 μL RNA respectively and 10 μL NASBA amplification reaction solution and heat at 65°C for 2 minutes, Then cool at 41°C for 2 minutes; immediately add 5 μL enzyme mixture and mix well, ddH 2 O to make up the reaction volume of 20 μL, react at 41°C for 90 min, stop the reaction in ice bath, and perform ELISA detection with NASBA product detection reagent.

[0080] As a result, only the RNA detection result of TB was positive, while the detection results of other pathogens were all negative, indicating that the kit was specific for the detection of TB RNA. (See Table 1)

[0081] Table 1 Specificity test of TB-NASBA kit

[0082] Table1 The specific test of TB-NASBA Kit

[0083]

example 2

[0084] Example 2: Sensitivity detection of Mycobacterium tuberculosis NASBA kit:

[0085] Extract the total RNA of TB virus, perform 10-fold serial dilution on RNA with an initial concentration of 1ng / μL TB, take 0.5μL template RNA and 10μL NASBA amplification reaction solution for each gradient, heat at 65°C for 2min, and cool at 41°C for 2min; Immediately Add 5 μL enzyme mixture and mix well, ddH 2 Make up 20 μL volume with O, react at 41°C for 90 minutes, stop the reaction in ice bath, and detect NASBA products by ELISA.

[0086] The results show that TB's RNA was diluted to l0 -5 There are still positive amplification products, and the sensitivity is 10fg, indicating that the kit has high sensitivity. (See Table 2).

[0087] Table 2 Sensitivity test of TB-NASBA kit

[0088] Table2 The sensitive test of TB-NASBA Kit

[0089]

example 3

[0090] Example 3: Detection of Mycobacterium deer tuberculosis NASBA-ELISA kit for clinical samples

[0091] (1) Specimen collection and preservation:

[0092] 28 deer lung samples of tuberculosis positive and 30 negative deer lung samples (from deer farms around Jilin Province) were collected by culture method.

[0093] (2) Preparation of sample RNA

[0094] (a) All plastic products involved in RNA extraction were sterilized with DEPC water combination, and the glassware was baked at 180°C for 6 hours to ensure that there was no RNase in the supplies, and the operation was performed on ice as much as possible.

[0095] (b) Grind the deer lung sample with 1ml 4% NaOH, incubate at 37°C for 30 minutes, shake once every 3 minutes during the period, centrifuge at 12000r / min for 5 minutes, remove the supernatant, add 1ml of sterilized deionized water to shake and mix well (repeat 3 times) Second-rate).

[0096](c) 4°C, 13000r / min, centrifuge for 8min, discard the supernatant and...

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Abstract

The invention relates to the technical field of biological detection, and is a rapid detection kit for Mycobacterium tuberculosis NASBA-ELISA in deer, which is characterized in that it consists of specific primers and capture probes designed according to the sequence of the conserved region of Mycobacterium tuberculosis 16srRNA gene. needle, detection probe, optimized NASBA amplification system and optimized NASBA product detection system. It can perform simple and rapid detection of Mycobacterium tuberculosis in deer, has the advantages of high specificity and easy promotion, and is also helpful for the early diagnosis of bovine and human multiple Mycobacterium tuberculosis; the detection method is designed and artificially Synthesize specific primers, capture probes and detection probes for the conserved region of the Mycobacterium tuberculosis gene sequence, determine the steps of the NASBA amplification reaction program and the NASBA product detection system, and can simultaneously monitor human and bovine multiple Mycobacterium tuberculosis, The detection result has high sensitivity and good repeatability.

Description

technical field [0001] The invention relates to the technical field of biological detection, and relates to a rapid detection kit and detection method of mycobacterium tuberculosis NASBA-ELISA in deer. Background technique [0002] Deer breeding is one of the pillar industries of China's special animal breeding. As traditional Chinese medicine, deer antler, antler plate, and deer fetus are increasingly widely used in traditional Chinese medicine treatment and health care. However, at present, the prevention and control of tuberculosis (TB) in deer herds has not been effectively controlled, which not only affects the economic output value of the vast farms, but also has an important impact on the prevention and control of human tuberculosis. As a zoonotic disease, deer tuberculosis is a chronic, consumptive infectious disease caused by Mycobacterium tuberculosis (M.tuberculosis), commonly known as Mycobacterium tuberculosis. It is caused by a Class B animal disease that must...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6804C12Q1/6865C12Q1/04
CPCC12Q1/6804C12Q1/6865C12Q2531/143C12Q2537/143
Inventor 徐亚维尹锐薛健于双孙亚娟王俊玲楚海娇王欢
Owner JILIN AGRI SCI & TECH COLLEGE
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