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Specific primer pair for assisting identification of Klebsiella pneumoniae

A Klebsiella, specific primer pair technology, applied in microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low sensitivity, cumbersome and complicated methods, easy to ignore, etc. Simple steps and increased accuracy

Inactive Publication Date: 2012-03-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this process, a large number of clones need to be selected for colony PCR and sequencing to detect all symbiotic bacteria. The method is cumbersome and complicated, and the sensitivity is not high, and it is easy to ignore certain types of symbiotic bacteria with low content.

Method used

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  • Specific primer pair for assisting identification of Klebsiella pneumoniae
  • Specific primer pair for assisting identification of Klebsiella pneumoniae
  • Specific primer pair for assisting identification of Klebsiella pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, the design of specific primer pair

[0032] 1. Acquisition of Klebsiella pneumoniae 16s rDNA sequence

[0033] Each B. dorsalis sample (sample 1 to sample 17) was identified as follows:

[0034] 1. Extraction of Bactrocera dorsalis genomic DNA

[0035] The blood / cell / tissue genomic DNA extraction kit of Beijing Tiangen Biochemical Technology Co., Ltd. was used to extract the genomic DNA of the whole body of a single adult worm.

[0036] 2. Use the universal primers of 16s rDNA sequence for PCR amplification

[0037] The general primers 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3') of the bacterial 16s rDNA sequence were used to amplify the 16s rDNA sequence of Bactrocera dorsalis symbiotic bacteria, and the length of the target amplification product was about 1500bp .

[0038] The total volume of PCR amplification is 50 μL, of which 5 μL 10×Reaction Buffer (containing Mg 2+ ), 4μL dNTPmixture (2.5mM), Taq enzyme 0.5μL (2.5U / ...

Embodiment 2

[0053] Embodiment 2, application-specific primer pair identification Klebsiella pneumoniae

[0054] 1. Genomic DNA of Klebsiella pneumoniae was extracted using a bacterial genomic DNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.).

[0055] 2. Using the genomic DNA extracted in step 1 (using water as a negative control) as a template, carry out PCR amplification with the specific primer pair designed in step 2 of Example 1 to obtain a PCR amplification product.

[0056] PCR amplification volume (50μL): 5μL 10×Reaction Buffer (containing Mg 2+ ), 4μL dNTP mixture (2.5mM), Taq enzyme 0.5μL (2.5U / μL, Beijing Tiangen Biochemical Technology Co., Ltd.), ddH 2 O 37.5 μL, genomic DNA 1 μL (115.8 ng / μL), upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL.

[0057] PCR amplification conditions: 94°C for 3min; 94°C for 30sec, 59.6°C for 30sec, 72°C for 30sec, 35 cycles; 72°C for 5min.

[0058] 3. Take 5 μL of the PCR amplification product, perform 1.5% aga...

Embodiment 3

[0060] Embodiment 3, application specific primer is identified the endosymbiotic Klebsiella pneumoniae of Bactrocera dorsalis

[0061] Each B. dorsalis sample (sample 1 to sample 17) was identified as follows:

[0062] 1. Genomic DNA extraction

[0063] Using the blood / cell / tissue genomic DNA extraction kit from Beijing Tiangen Biochemical Technology Co., Ltd., the genomic DNA of the whole body of a single adult Bactrocera dorsalis was extracted.

[0064] 2. PCR identification

[0065] Using the genomic DNA extracted in step 1 as a template (with water as a negative control), PCR amplification was performed with the specific primer pair designed in step 2 of Example 1 to obtain a PCR amplification product.

[0066] PCR amplification volume (50μL): 5μL 10×Reaction Buffer (containing Mg 2+ ), 4μL dNTP mixture (2.5mM), Taq enzyme 0.5μL (2.5U / μL, Beijing Tiangen Biochemical Technology Co., Ltd.), ddH 2 O 37.5 μL, genomic DNA 1 μL (115.8 ng / μL), upstream primer (10 μM) 1 μL, do...

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Abstract

The invention discloses a specific primer pair for assisting identification of Klebsiella pneumoniae. The primer pair provided in the invention consists of DNA shown in sequence 1 of the sequence table and DNA shown in sequence 2 of the sequence table. The specific primer pair designed in the invention can be used for assisting identification about whether Bactrocera dorsalis (Hendel) carries Klebsiella pneumoniae only by means of PCR (polymerase chain reaction) and electrophoresis, and the identification is characterized by high sensitivity, simple process and no need for cloning. In order to increase accuracy, a PCR amplified product can be subjected to sequencing. The specific primer pair provided in the invention improves the efficiency of Bactrocera dorsalis (Hendel) symbiotic bacteria (Klebsiella pneumoniae), and creates conditions for further study of the coevolution relation between Bactrocera dorsalis (Hendel) and Klebsiella pneumoniae.

Description

technical field [0001] The invention relates to a pair of specific primers for auxiliary identification of Klebsiella pneumoniae. Background technique [0002] Bactrocera orange (Bactrocera) dorsalis (Hendel), also known as oriental fruit fly, belongs to Diptera (Diptera), Tephritidae (Tephritidae), Bactrocera (Bactrocera). The insect has a wide range of hosts and can harm more than 40 families and more than 250 kinds of fruits and vegetables such as citrus, guava, carambola, etc. It is a worldwide quarantine pest. Bactrocera dorsalis invaded my country from abroad, spread from south to north in the country, and the trend of further spread became increasingly serious, posing a greater threat to the export of my country's main fruit products. At present, Bactrocera dorsalis is mainly distributed in Guangdong, Guangxi, Hunan, Guizhou, Fujian, Hainan, Yunnan, Sichuan, Taiwan and other provinces. The wide distribution and serious harm to my country's agricultural and forestry ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/22
Inventor 李志红柳丽君
Owner CHINA AGRI UNIV
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