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Double PCR (Polymerase chain reaction) primer for simultaneous detection of Klebsiella pneumoniae and Aeromonas caviae and detection method of double PCR primer

A technology for Aeromonas guinea pigs and Klebsiella, which is used in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc. Complex problems, to achieve the effect of quick start, strong promotion, and simple operation

Inactive Publication Date: 2017-05-24
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have the advantages of being unaffected by the course of the disease, rapid, high sensitivity and specificity, and novel, but they are costly, require high experimental conditions, require sophisticated and expensive instruments, and are complicated to operate, so they are not suitable for detection and promotion at the grassroots level.

Method used

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  • Double PCR (Polymerase chain reaction) primer for simultaneous detection of Klebsiella pneumoniae and Aeromonas caviae and detection method of double PCR primer
  • Double PCR (Polymerase chain reaction) primer for simultaneous detection of Klebsiella pneumoniae and Aeromonas caviae and detection method of double PCR primer
  • Double PCR (Polymerase chain reaction) primer for simultaneous detection of Klebsiella pneumoniae and Aeromonas caviae and detection method of double PCR primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 Klebsiella pneumoniae and Aeromonas caviae double PCR primers obtain

[0034] According to the Klebsiella pneumoniae ompC sequence (Genbank accession number KJ579291) and Aeromonas caviae ahal sequence (Genbank accession number JQ946880) that have been collected on the NCBI website, the online website Primer-BLAST ( https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) design two groups of specific primers, the nucleotide sequences of the primers are as follows:

[0035] Primer ompC:

[0036] Forward primer F: 5'-AACACTGAAAGCTCCAGCGA-3'

[0037] Reverse primer R: 5'-CGTCGGTACGTTTGGAGTGA-3';

[0038] Primer ahal:

[0039] Forward primer F: 5'-AGACGGTACCACTTTCGACG-3'

[0040] Reverse primer R: 5'-ATGTGACGCGGGTCTATGTC-3';

Embodiment 2

[0041] The specificity detection of embodiment 2 Klebsiella pneumoniae and Aeromonas caviae double PCR primers

[0042] 1. Genomic DNA extraction of Klebsiella pneumoniae, Escherichia coli, Edwardsiella tarda, Aeromonas caviae, Aeromonas hydrophila, and Aeromonas victoria.

[0043] Bacterial strain DNA extraction: use Ezup Column Bacterial Genomic DNA Extraction Kit (Shanghai Sangong) manual guide (the public can obtain detailed information from Shanghai Sangong), as shown in Table 1, DNA dissolved in 50 μL, pH7 .5 in TE buffer and stored at -20°C for later use.

[0044] Table 1

[0045]

[0046]

[0047] 2. Double PCR reaction system and procedure:

[0048] The total volume of the double PCR detection system is 25 μL, including:

[0049] ompC and ahal upstream and downstream primers each 0.5 μL (100mmol / L)

[0050] 10×Buffer (including Mg 2+ )3μL

[0051] dNTP 2μL (25g / L)

[0052] Taq enzyme 0.2 μL

[0053] 3 μL of each DNA template

[0054] Finally, make up to 2...

Embodiment 3

[0060] Example 3 Sensitivity detection of Klebsiella pneumoniae and Aeromonas caviae double PCR method

[0061] Through Klebsiella pneumoniae ompC sequence and Aeromonas caviae ahal sequence gene cloning and concentration adjustment, the highest DNA concentration of Klebsiella pneumoniae and Aeromonas caviae were both 2.5×10 5 pg / μL, and then prepared by 3-fold serial dilution until 3.3×10 pg / μL. Carry out by adding other PCR reaction reagents except the DNA template in Example 1, and the amplified product is detected by electrophoresis.

[0062] The results of the double PCR sensitivity test were as follows: figure 2 As shown, among them, M: DL2000 DNA Marker; 1~9 are respectively: Klebsiella pneumoniae DNA concentration is 2.5×10 5 pg / μL, 8.3×10 4 pg / μL, 2.8×10 4 pg / μL, 9.3×10 3 pg / μL, 3.1×10 3 pg / μL, 1.0×10 3 pg / μL, 3.3×10 2 pg / μL, 1.1×10 2 pg / μL, 3.3×10 pg / μL; DNA concentrations of Aeromonas caviae were 2.5×10 5 pg / μL, 8.3×10 4 pg / μL, 2.8×10 4 pg / μL, 9.3×10 3...

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Abstract

The invention relates to a double PCR (Polymerase chain reaction) primer for simultaneous detection of Klebsiella pneumoniae and Aeromonas caviae and a detection method of the double PCR primer, and belongs to the technical field of biological detection. The Klebsiella pneumoniae and Aeromonas caviae can be simultaneously detected in the same reaction system by taking a Klebsiella pneumoniae ompC gene and an Aeromonas caviae ahal gene as detection genes, the sizes of amplified products are 418bp and 213bp respectively, and the detection sensitivity of the two pairs of primers is 1.0*10<3>pg / mu L and 3.3*10<2>pg / mu L respectively. The double PCR detection method provided by the invention has the advantages of high specificity, high sensitivity, simple operation and short time, and can be used for simultaneous detection of Klebsiella pneumoniae and Aeromonas caviae.

Description

technical field [0001] The invention relates to a double PCR primer and a detection method for synchronous detection of Klebsiella pneumoniae and Aeromonas caviae, belonging to the technical field of biological detection. Background technique [0002] Klebsiella pneumoniae (Klebsiella Pneumoniae) belongs to Enterobacteriaceae (Enterobacteriaceae) Klebsiella (Klebsiella), is a capsuled Gram-negative bacteria, including subsp.pneumoniae (subsp.pneumoniae), rhinitis subspecies (subsp.azaenae) and nasal induration subspecies (subsp.rhinoscleromatis) 3 subspecies. Klebsiella pneumoniae was first isolated in 1882 and is second only to Escherichia coli as an opportunistic pathogen in Gram-negative bacterial infections. It is a zoonotic pathogen that mainly causes pneumonia, respiratory diseases, enteritis, sepsis, etc. in humans. Researchers have successively isolated and discovered this bacteria from carps, mice, sika deer, cattle, giant pandas and other animals. Due to the redu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 滕涛习丙文梁利国陈凯潘良坤谢骏徐跑
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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