Identification method for Klebsiella pneumoniae and adopted primers

A Klebsiella, identification method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve the problems of complex quantitative measurement instruments, lack of laboratory equipment, high cost, etc. Achieve the effect of simple test steps, improve detection efficiency and save time

Active Publication Date: 2015-04-22
王贵升
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to distinguish Klebsiella pneumoniae from Escherichia coli in conventional bacterial culture, and it takes a long time for bacterial biochemical identification and 16sRNA identification, about 3-4 days
If 16sRNA identification needs to be sequenced, the cost is relativ

Method used

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  • Identification method for Klebsiella pneumoniae and adopted primers
  • Identification method for Klebsiella pneumoniae and adopted primers
  • Identification method for Klebsiella pneumoniae and adopted primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Artificially synthesized primer pair K1, K2;

[0039] K1:

[0040] K1-F is: 5-CGATGCTACTTATCCCGACA-3, as shown in SEQ ID NO.1,

[0041] K1-R is: 5-ACCACCAGCAGACGAACTT-3, as shown in SEQ ID NO.2;

[0042] K2:

[0043] K2-F is: 5-CGGAGCGTTTTTCAATCGG-3, as shown in SEQ ID NO.3,

[0044] K2-R is: 5-TGAGCGGGTAATAAATGCGG-3, as shown in SEQ ID NO.4.

Embodiment 2

[0046] Recover Klebsiella pneumoniae on ordinary nutrient agar, pick a single colony, shake the bacteria overnight, and use the bacterial solution as a template for PCR reaction. PCR reaction system: 2×Taq PCR MasterMix 25 μL, K1-F and K1-R (upper and lower primers) 2 μL each (or K2-F and K2-R 2 μL each), use bacterial liquid as template 4 μL, and finally use sterilized double steamed Water was added to 50 μL. The reaction conditions are: first step, pre-denaturation at 94°C for 5 minutes; second step, denaturation at 94°C for 30 seconds; third step, annealing at 58°C for 30 seconds; fourth step , extended at a temperature of 72° C. for 1 min; from the second step to the fourth step, a total of 35 cycles were performed; the fifth step: a final extension at a temperature of 72° C. for 7 min; the PCR amplification ended. The amplified product was sequenced, and its sequence was shown in SEQ ID NO.5, which was the specific gene sequence of Klebsiella pneumoniae. It can be...

Embodiment 3

[0051] Specificity test for Escherichia coli

[0052] The 10 strains of Escherichia coli stored in our laboratory were subjected to PCR amplification reaction using the method in Example 2 (the template for PCR is a single colony). After the PCR amplification reaction, 5ul PCR amplification products were taken and subjected to 1.5% (W / V) agarose gel electrophoresis, and the electrophoresis results were photographed on the BIO-RAD ultraviolet gel imaging system; the results are as follows: figure 2 As shown, no band appeared at 303bp. It can be seen that this primer cannot detect Escherichia coli.

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Abstract

The invention relates to an identification method for Klebsiella pneumoniae and adopted primers, and belongs to the field of biotechnology. The two pairs of primers (artificially synthesized gene sequences) are acquired through creative labor and used for conducting PCR amplification on the Klebsiella pneumoniae, escherichia coli and pseudomonas aeruginosa, and an amplified product is a specificity sequence of the Klebsiella pneumoniae. Thus, the Klebsiella pneumoniae can be distinguished from the escherichia coli and the pseudomonas aeruginosa. The primers for identifying the Klebsiella pneumoniae comprise the primer pair K1 or/and the primer pair K2, wherein the primer pair K1 comprises K1-F and K1-R, and the primer pair K2 comprises K2-F and K2-R. Nucleotide sequences of K1-F, K1-R, K2-F and K2-R are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 in a sequence table.

Description

technical field [0001] The invention relates to a method for identifying Klebsiella pneumoniae and primers used, belonging to the field of biotechnology. Background technique [0002] Klebsiella pneumoniae (K.pnenmoniae) is a G-bacteria belonging to the Enterobacteriaceae family and is an opportunistic pathogen. Klebsiella pneumoniae and Escherichia coli have a high similarity on ordinary nutrient agar, MacConkey agar and other media. The shape, color and smell of the bacteria are very similar, and they are all short in the Gram staining microscope. G-microbacteria. It is difficult to distinguish Klebsiella pneumoniae from Escherichia coli in conventional bacterial culture, and it takes a long time, about 3-4 days, to do the biochemical identification of bacteria and the identification of 16sRNA. If 16sRNA identification needs to be sequenced, the cost is relatively high. Although the fluorescent real-time quantitative polymerase chain reaction (real time PCR) technology...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12R1/22
CPCC12Q1/689
Inventor 王贵升张婷婷田夫林王金宝单虎
Owner 王贵升
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