Fluorescent quantitative loop-mediated isothermal amplification (LAMP) detection method for bovine tuberculosis pathogene

A fluorescence quantitative and tuberculosis technology, applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of expensive reagents, long detection time, lack of specificity, etc., and achieve curative effect judgment and prognosis The effect of simple evaluation, various methods of judging results, and accurate analysis of results

Inactive Publication Date: 2012-11-21
CHINESE ACAD OF INSPECTION & QUARANTINE
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Problems solved by technology

Although the bacteriological examination method is simple in equipment and easy to operate, it cannot accurately identify Mycobacterium tuberculosis and atypical mycobacteria, lacks specificity, and takes a long time; although immunological examination is simple and easy to perform, due to the expensive reagents at present, Leading to high cost of large-scale implementation, long detection time and other problems

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  • Fluorescent quantitative loop-mediated isothermal amplification (LAMP) detection method for bovine tuberculosis pathogene
  • Fluorescent quantitative loop-mediated isothermal amplification (LAMP) detection method for bovine tuberculosis pathogene
  • Fluorescent quantitative loop-mediated isothermal amplification (LAMP) detection method for bovine tuberculosis pathogene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The construction of embodiment 1 standard plasmid

[0048] 1.1 Amplification of the target gene

[0049] Using the Mycobacterium bovis genomic DNA as a template and using SEQ ID No.7 and SEQ ID No.8 as primers, the target fragment of the IS1081 sequence is amplified. Add the following reagents to a 0.2ml PCR tube:

[0050] Table 2 Common PCR reaction system (50μL)

[0051]

[0052] After mixing, put it into a PCR amplification instrument and perform amplification according to the following procedures:

[0053] The cycle parameters of IS1081 sequence amplification were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min, annealing at 60.8°C for 1 min, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min.

[0054] After the reaction, the fragment size of the product was identified by 0.8% agarose gel electrophoresis. Compared with the DNA Marker, if it was consistent with the target fragment size defined by the primers, subsequent exp...

Embodiment 2

[0102] Copy number=plasmid concentration (g / μl)×injection volume×Arbeck’s constant / (total plasmid length×average molecular weight of one base pair), and finally the plasmid was diluted 10 times with Tris-Hcl (10mM). By calculating the copy number of the plasmid extracted by the present invention is 2.76×10 10 copy, according to the copy number, the plasmid was diluted to 1×10 10 copy / μL, and then perform a 10-fold dilution. The establishment of embodiment 2 bovine tuberculosis pathogen LAMP method

[0103] Take 1 μL of the positive plasmid DNA extracted in Example 1 as a template, configure the reaction system according to the following system, and replace the plasmid DNA template with sterile water as a negative control. When the concentration of one of the components is optimized, the other components remain unchanged, and the volume of the changed component can be equilibrated with double distilled water.

[0104] Table 4 Fluorescence quantitative LAMP reaction system (2...

Embodiment 3

[0162] The DNA extracted from 24 bovine tuberculosis positive blood samples was detected by the established fluorescent quantitative LAMP method according to the established method in Example 2. The result is as Figure 13 shown. The results showed that the positive rate was 100%, consistent with the expected effect. prove the feasibility of this method.

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Abstract

The invention discloses a fluorescent quantitative loop-mediated isothermal amplification (LAMP) detection method for bovine tuberculosis pathogene. The method comprises the following steps of: performing LAMP amplification on an insertion sequence IS1081 of a mycobacterium composite by internal primers shown in SEQ ID No.1 and SEQ ID No.2, external primers shown in SEQ ID No.3 and SEQ ID No.4 and loop primers shown in SEQ ID No. 5 and SEQ ID No.6 and by taking blood DNA of cattle to be detected as a template, a positive standard substance as positive control and sterile water as negative control, and collecting fluorescent signals. The method is high in sensitivity and specificity and is easy and quick to operate, and results are accurate.

Description

technical field [0001] The invention belongs to the field of animal molecular biology, in particular to a method for detecting bovine tuberculosis pathogen fluorescence quantitative LAMP. Background technique [0002] Bovine tuberculosis is listed as a B-type animal disease by the World Organization for Animal Health (OIE), and it is listed as a B-type animal disease in my country. According to the statistics of the World Health Organization, from 1986 to 1990, 41.5% of developing countries and 25% of developed countries had tuberculosis epidemics on the rise. Currently, bovine tuberculosis has been controlled in many developed countries. But it still threatens the health of hundreds of millions of people in many developing countries. As early as 1960, the Seventh Meeting of the World Health Organization Expert Committee reported that when analyzing the factors affecting the prevalence of tuberculosis, it pointed out: "Unless bovine tuberculosis is eradicated, the control ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 贾广乐林祥梅韩雪清王慧煜
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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