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PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis

A technology of Mycobacterium bovis and Mycobacterium, which is applied in the field of PCR identification, can solve the problems of unfavorable clinical test samples and high economic cost, achieve simple operation, high-throughput diagnosis and detection, and reduce false negative or false positive results. effect of probability

Inactive Publication Date: 2012-04-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned methods only separately provide PCR-related methods for identifying a certain type of bacteria, and some use methods with high economic costs such as probe hybridization and fluorescence quantification. A report on the identification of major bacteria of the genus Mycobacteria using a complete set of primers

Method used

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  • PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis
  • PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis
  • PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis

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Experimental program
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Effect test

Embodiment 1

[0056] The design of embodiment 1PCR specific primer

[0057] The 16S rDNA gene corresponding to 16SrRNA is highly conserved in the bacterial genome, and can identify the principle of bacteria of the genus. Firstly, primers are designed according to the 16Sr RNA conserved region of Mycobacterium. The target fragment size is 543bp sequence, which is designed using software and Primer 3 Primer, the primer sequence is:

[0058] 16SrRNA-F 5'-ACGGTGGGTACTAGGTGTGGGTTTC-3';

[0059] 16SrRNA-R 5'-TCTGCGATTAGCGACTAAGACTTCA-3';

[0060] Rv0577 is a restricted gene of MTBC, primers were designed according to this gene, and the size of the target fragment amplified by PCR was 786bp. The primer sequences are:

[0061] MTBC-F 5'-ATGCCCAAGAGAAGCGAATACAGGCAA-3';

[0062] MTBC-R 5'-CTATTGCTGCGGTGCGGGCTTCAA-3';

[0063] Since the deleted region 7 (deleted region7, RD7) does not exist in other MTBC strains, only Mycobacterium tuberculosis has this gene region, and primers are designed accor...

Embodiment 2

[0072] The establishment of embodiment 2PCR amplification method

[0073] 1. The PCR reaction system uses the total bacterial DNA as a template for PCR reaction. The 20 μL reaction system is:

[0074]

[0075] 2. PCR reaction conditions

[0076] The reaction program of PCR containing 16SrRNA, MTBC, PncA primer pair is: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 40s, annealing at 60°C for 40s, extension at 72°C for 40s, 31 cycles, and finally extension at 72°C for 10 minutes;

[0077] The reaction program of PCR containing MT primer pair is: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 40s, annealing at 61°C for 40s, extension at 72°C for 40s, 31 cycles, and finally extension at 72°C for 10 minutes;

[0078] The reaction program of PCR containing the RD1 primer pair was: pre-denaturation at 94°C for 5 min, followed by denaturation at 94°C for 40 s, annealing at 62°C for 40 s, extension at 72°C for 40 s, 31 cycles, and fina...

Embodiment 3

[0080] The specificity test of embodiment 3PCR method

[0081]Mycobacterium tuberculosis C68503, Mycobacterium bovis C68001, Mycobacterium bovis BCGC68017, Mycobacterium smegmatis cm 2 155. The DNA of Mycobacterium avium C68201, Staphylococcus CAU0411, and Escherichia coli CAU0439 was used as a template, and water was used as a blank control. 5 pairs of primers of the present invention were used respectively, and the PCR method established by the present invention was used for inspection. After the PCR reaction ended, the agar Judgment results of specific amplification bands by sugar gel electrophoresis.

[0082] test results:

[0083] PCR results of 16SrRNA primers: the PCR amplification products of Mycobacterium were positively amplified, while the other strains and the blank control had no target amplified fragments. For the results, see figure 1 .

[0084] PCR results of MTBC primers: positive amplification of Mycobacterium tuberculosis, Mycobacterium bovis and BCG, bu...

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Abstract

The invention provides five pairs of primers and an identification method for PCR(Polymerase Chain Reaction) identification of mycobacterium bovis. The five pairs of primers respectively amplify aiming at a 16SrRNA conserved region of mycobacterium bacteria, a mycobacterium tuberculosis complex(MTBC)Rv0577 gene, Rv1970 of a mycobacterium tuberculosis RD7 region, a mycobacterium bovis pncA gene and an RD1 region gene to generate specific amplified fragments, and the nucleotide sequences of the specific amplified fragments are as shown in SEQ ID No.1-5. According to the identification method provided by the invention, the total DNA of a sample is taken as a template, PCR amplification is respectively performed by the five pairs of primers, and the result is judged according to the size of an amplified band. The primers provided by the invention can specifically identify mycobacteria, MTBC, mycobacterium tuberculosis, mycobacterium bovis and mycobacterium bovis BCG(Bacillus Calmette-Guerin), and the detection method has good sensitivity and simplicity of method and operation, and can realize quick large-flux detection of mycobacterium bovis.

Description

technical field [0001] The invention relates to biological detection and identification technology, in particular to a primer for PCR identification of mycobacterium bovis and a PCR identification method using the primer. Background technique [0002] Bovine tuberculosis is mainly a chronic wasting infectious disease caused by Mycobacterium bovis, which can be transmitted to humans or other animals through sick animals or their products. Most cattle infected with Mycobacterium bovis are not overtly infected or have clinical symptoms only in the late stage, so early clinical diagnosis is not easy to obtain accurate results. Pathological examination is one of the most reliable methods. Pathological examination can be confirmed based on characteristic lesions, but it must be carried out after the animal is dead or slaughtered, and the required diagnosis period is long, which takes 3-8 weeks, so it is often not used in actual work . Since milk, beef and other foods are closely...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/32
Inventor 周向梅赵德明李华林敬钧杨利峰尹晓敏杨春华
Owner CHINA AGRI UNIV
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