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Primers and probes for quantitative detection of Klebsiella pneumoniae and their application

A technology for quantitative detection of Klebsiella, applied to primers and probes for quantitative detection of Klebsiella pneumonia and its application field, can solve the problems of long detection period, low sensitivity and low accuracy.

Active Publication Date: 2015-09-09
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, domestic food sanitation microbial inspection methods mainly adopt national standards (GB / T) and industry standards (SN / T). Such standards are mainly composed of traditional isolation culture, microscopic observation, biochemical identification and other experiments, and the operation steps are complicated. , the workload is heavy, and it takes 5-8 days from cultivation to biochemical identification. At the same time, the detection accuracy is low, which is far from meeting the requirements of modern detection.
Clinical testing requires tests such as sputum culture or blood culture, and the testing cycle is long, which cannot meet the needs of treatment
Conventional PCR technology currently has disadvantages such as cumbersome operation and easy cross-contamination leading to false positives.
Immunological detection technology has the advantages of fast, simple and low cost, but requires high-quality and high-stability monoclonal antibodies. Currently, it has disadvantages such as poor specificity, low sensitivity, and low accuracy, and can only be used as an auxiliary detection method.
The loop-mediated constant temperature amplification technology that has emerged in recent years has the advantages of simple, fast, high sensitivity, and does not rely on expensive equipment. It is very suitable for use in grassroots laboratories, but there are difficulties in primer design, poor accuracy and sensitivity, and false positives Disadvantages such as high rate and immature market

Method used

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  • Primers and probes for quantitative detection of Klebsiella pneumoniae and their application
  • Primers and probes for quantitative detection of Klebsiella pneumoniae and their application
  • Primers and probes for quantitative detection of Klebsiella pneumoniae and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Preparation of RecA gene sequence standard:

[0024] In order to establish a real-time fluorescent quantitative PCR method, the external standard required by the method must be prepared first. The standard should contain highly conserved and specific sequences, and high specificity of the reaction should be ensured. The present invention uses the conserved region of the Klebsiella pneumoniae RecA gene sequence as the target sequence. Mainly use PCR technology to amplify the RecA gene sequence of Klebsiella pneumoniae, use gene recombination technology to connect it to the plasmid vector pMD18-T, construct the recombinant plasmid pMD18-T-KpRecA, and carry out corresponding PCR identification and sequencing identification , and finally quantified as a standard for the method to be established, laying the foundation for the next method and evaluation.

[0025] 1. Preparation of template DNA:

[0026] 1. Extract Klebsiella pneumoniae genomic DNA and use it as a template f...

Embodiment 2

[0067] Preliminary establishment of real-time fluorescent quantitative PCR detection method for Klebsiella pneumoniae:

[0068] 1. Design and synthesis of specific primers and probes:

[0069] Taking the conserved fragment of the ITS sequence of Klebsiella pneumoniae selected above as the target, a set of real-time fluorescent quantitative PCR primers and probes were designed using Primer express3 software, Primer Premier5 software and Oligo7 software.

[0070] As the core of the present invention, a group of primers and probe nucleotide sequences for Klebsiella pneumoniae real-time fluorescent PCR detection are as follows:

[0071] Upstream primer: KprecA19F: 5'-TTCTTCTGCGTCGTTGCC-3',

[0072] Downstream primer: KprecA144R: 5'-GCGATCACCTGGCTGAAAG-3'.

[0073] Probe: KprecA51P: 5'-FAM-TCTGGCTTCGCATCCTGATTGTTGA-TAMRA-3'.

[0074] The primers and probes amplify the target nucleotide sequence as:

[0075] 5'-TTCTTCTGCGTCGTTGCCGTCAACAACGAAGTCTGGCTTCGCATCCTGATTGTTGAGCAGCAGTTCGC...

Embodiment 3

[0089] Evaluation experiment:

[0090] 1. Sensitivity experiment:

[0091] The plasmid prepared above was diluted 10 times to obtain a series of concentrations of the plasmid, and the selected concentration was

[0092] 1.00×108 copies / ml, 1.00×10 7 copies / ml, 1.00×10 6 copies / ml, 1.00×10 5 copies / ml,

[0093] 1.00×10 4 copies / ml, 1.00×10 3 copies / ml, 1.00×10 2 copies / ml etc. as gradient templates. The reaction system is shown in Table 4.

[0094] Table 4

[0095] Element

volume

2×premix

10μl

Primer F (5uM)

1.5μl

Primer R (5uM)

1.5μl

Probe (5uM)

0.75μl

template

2μl

wxya 2 o

Make up to 20μl

[0096] The reaction conditions were: 94°C pre-denaturation for 2 minutes, 94°C for 15 seconds, 60°C for 40s and collecting fluorescence signals, 40 cycles. According to the fluorescence signal detected by the instrument, the fluorescence curve is obtained by software processing, and the sign...

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Abstract

The invention discloses a primer and a probe for quantitative determination of klebsiella pneumonia, and an application of the primer and the probe. Nucleotide sequences of the primer are a sequence 3 and a sequence 4 in a sequence table; a nucleotide sequence of the probe is a sequence 5 in the sequence table. The primer and the probe have the beneficial effects that the specific primer and probe sequences of the klebsiella pneumonia disclosed by the invention can be applied to qualitative and quantitative detection of the klebsiella pneumonia, the target of accurately and quantitatively determining the deoxyribonucleic acid (DNA) content of the klebsiella pneumonia in a specimen to be detected can be achieved by extracting the DNA in the sample to be detected and combining with a real-time fluorescence quantification polymerase chain reaction (PCR) detection technology, and the primer and the probe can be applied to food detection, scientific research and clinical diagnosis and used for carrying out qualitative and quantitative analysis on the klebsiella pneumonia DNA in samples such as a food sample, a suffer throat swab, nasopharyngeal secretion, people sputum specimens and blood samples. Thus, the primer and the probe have an important significance on judgment of klebsiella pneumonia infection, evaluation of treatment effectiveness and dynamic observation of the state of an illness, and simultaneously play an important role in the field of detection of clinical medicine.

Description

technical field [0001] The invention relates to a primer and a probe for quantitative detection of Klebsiella pneumoniae and application thereof. Background technique [0002] Klebsiella pneumoniae (Klebsiella pneumoniae) belongs to the Gram-negative Enterobacteraceae (Enterobacteraceae) Klebsiella (Klebsiella) genus, divided into three subspecies: pneumonia subspecies (subsp.pneumoniae), rhinitis subspecies ( subsp.azaenae) and subsp.rhinoscleromatis. Widely distributed in nature, it is an opportunistic pathogenic bacterium in the intestinal tract and respiratory tract of humans and animals. When the body’s immunity is reduced or long-term use of antibiotics in large quantities leads to bacterial flora imbalance, it can cause infection, which can cause pneumonia, bronchitis, sepsis, meningitis, liver disease, etc. Abscess, endophthalmitis, urinary system inflammation, or wound infection, etc., if not treated properly, the mortality rate is extremely high. Currently, it is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2545/113C12Q2563/107
Inventor 车团结李琳尤崇革
Owner SUZHOU BAIYUAN GENT CO LTD
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