PCR (Polymerase Chain Reaction) amplification primer for quickly detecting klebsiella pneumonia and application thereof

A Klebsiella and amplification primer technology, applied in the field of animal bacteriology and molecular biology, can solve the problems of long time consumption and insensitive detection, and achieve the effects of less time consumption, high sensitivity and low cost

Inactive Publication Date: 2018-09-14
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These techniques are not only time-consuming, but also insensitive to detection, while the PCR method can not only quickly and sensitively diagnose the pathogen, but also has stronger specificity for pathogenic infection in the incubation period

Method used

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  • PCR (Polymerase Chain Reaction) amplification primer for quickly detecting klebsiella pneumonia and application thereof
  • PCR (Polymerase Chain Reaction) amplification primer for quickly detecting klebsiella pneumonia and application thereof
  • PCR (Polymerase Chain Reaction) amplification primer for quickly detecting klebsiella pneumonia and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Establishment of a PCR method for rapid detection of Klebsiella pneumoniae

[0038] 1. Preparation of materials

[0039] Klebsiella pneumoniae, Cryptobacterium cryptica, Mannella hemolyticus, Pasteurella, Mycoplasma bovis, bovine parainfluenza virus type 3, bovine infectious rhinotracheitis virus and Escherichia coli were isolated, identified and preserved by Guangxi Veterinary Research Institute. Samples were obtained from veterinary clinics. 10×PCR Buffer, dNTPs, ES-Taq DNA polymerase, DNA / RNA extraction kit, bacterial genome DNA extraction kit were purchased from Kangwei Century Biotechnology Co., Ltd.

[0040] 2. Design and synthesis of PCR primers

[0041] According to the homology comparative analysis of the phoE gene sequence of Klebsiella pneumoniae in GenBank, the conserved sequence region was selected as the amplification region, and specific amplification primers were designed by using Oligo 7.0 primer design software and BLAST software program. ...

Embodiment 2

[0072] Example 2 Rapidly detects the annealing temperature test of Klebsiella pneumoniae PCR method

[0073] Perform PCR amplification at annealing temperatures of 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C, and 62 °C to determine the optimal annealing temperature. The results showed that the designed primers had a large tolerance to annealing temperature, and could amplify well under the reaction programs with annealing temperatures of 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃ and 62 ℃. Add a single destination strip ( figure 1 ). To this end, the reaction program used in this PCR method was: 95°C for 5 min; followed by 35 cycles of 95°C for 35 s, 58°C for 40 s, and 72°C for 45 s; and finally 72°C for 10 min.

Embodiment 3

[0074] Embodiment 3 detects the specific detection result of Klebsiella pneumoniae PCR method

[0075] Genomic DNA / RNA (RNA for reverse reaction) was extracted from Klebsiella pneumoniae, Cryptobacterium, Mannella hemolyticus, Pasteurella, Mycoplasma bovis, Bovine parainfluenza virus type 3, Bovine infectious rhinotracheitis virus and Escherichia coli. Transcribed into cDNA), using the optimized reaction system and reaction program for PCR amplification, testing the specificity of the detection method of the present invention, the results showed that only the Klebsiella pneumoniae sample amplified the target fragment band, for Positive results, no amplified bands were detected in the reaction tubes of the 7 strain control strains and the water control reaction tube, which was a negative result ( figure 2 ), indicating that the method has good specificity.

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Abstract

The invention discloses a PCR (Polymerase Chain Reaction) amplification primer for quickly detecting klebsiella pneumonia and application thereof, and belongs to the field of animal bacteriology and molecular biology. The PCR amplification primer is prepared from an upstream primer having a nucleotide sequence as shown in SEQ ID NO:1 and a downstream primer having a nucleotide sequence as shown inSEQ ID NO:2; the PCR amplification primer is used for preparing a PCR amplification kit for detecting the klebsiella pneumonia. A PCR detecting method provided by the PCR amplification kit has high specificity and sensitivity, good repeatability and high credibility and is capable of specifically detecting the klebsiella pneumonia and quickly and accurately obtaining a detecting result; meanwhile, the PCR amplification kit is low in cost and simple in operation, is suitable for being used at the primary level and can be used as a quick, accurate and simple detecting tool for quick laboratoryidentification of the klebsiella pneumonia and large-scale epidemiological investigation.

Description

technical field [0001] The invention relates to the technical fields of animal bacteriology and molecular biology, in particular to a rapid, simple and low-cost PCR amplification primer for detecting Klebsiella pneumoniae and its application. Background technique [0002] Klebsiella pneumonia (Klebsiella pneumonia, Kpn) is a Gram-negative bacterium belonging to the genus Klebsiella in the Enterobacteriaceae family. It can invade the intestines, respiratory tract and other organs of humans and animals, which can cause symptoms such as pneumonia, liver abscess, wound infection and sepsis, and is a zoonotic pathogen. At the same time, after cattle were infected with Klebsiella pneumoniae, their susceptibility to pathogens such as Pasteurella multocida, Mannella hemolyticus and Mycoplasma bovis increased. At present, the pathogen is distributed all over the world, and it is also widely distributed in China, causing huge economic losses and becoming one of the important pathogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12N15/11C12R1/22
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 李军吴翠兰彭昊潘艳冯世文陶立李常挺马春霞谢永平钟舒红胡帅贺会利
Owner GUANGXI VETERINARY RES INST
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