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Klebsiella pneumoniae characteristic sequence and qPCR rapid detection system and method thereof

A Klebsiella pneumoniae, characteristic sequence technology, which is applied in biochemical equipment and methods, microorganism-based methods, and microbial determination/inspection, etc. Lack of practical guidance and other issues to achieve the effect of improving clinical treatment effect, specificity, and reducing false negative and false positive rates

Active Publication Date: 2021-04-30
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In the existing patent literature, CN111549150A, CN110423837A, CN110499374A, CN105624284A, CN105950772A, etc. have all recorded detection methods for Klebsiella pneumoniae, but none of these detection methods involves the detection of clinical samples, and lacks practical guidance; in addition, currently None of the existing detection methods can effectively solve the problem of false positive and high false negative rates in the detection process of clinical samples

Method used

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  • Klebsiella pneumoniae characteristic sequence and qPCR rapid detection system and method thereof
  • Klebsiella pneumoniae characteristic sequence and qPCR rapid detection system and method thereof
  • Klebsiella pneumoniae characteristic sequence and qPCR rapid detection system and method thereof

Examples

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Effect test

Embodiment 1

[0032] K.pneumoniae RJF999, K.pneumoniae HS11286, E.coli M15, E.coli DH5α, P, aeruginosa PAO1, S.enterica ATCC 9150, S.enterica H9812, A.baumanni lac4, A.baumanni A19 were tested using the detection system of the present invention -29, K.oxytoca 333, a total of 6 kinds of 10 standard strains were tested.

[0033] 1. Experimental bacterial culture

[0034] This example involves standard strains and clinical isolates that are frozen and preserved according to conventional bacteria (see: J. Sambrook, D.W. Russell. Molecular Cloning Experiment Guide, 2002, Beijing: Science Press), and the strains are plated at 37°C Cultivate for 24 hours, pick a single colony, inoculate an appropriate amount of liquid medium, and continue to cultivate at 200 rpm for 16-24 hours. Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus were cultured in LB solid or liquid medium; Salmonella was cultured in TSB (liquid) or ...

Embodiment 2

[0069] From the clinical isolates of Klebsiella pneumoniae isolated in this study, 9 strains were randomly selected and detected by the detection method of the present invention. The qPCR reaction system and reaction procedure are the same as in Example 1, and the detection results are shown in Figure 5 .

[0070] from Figure 5 It can be seen that, with Ct=20 as the cutoff value, the Ct values ​​of all clinical strain amplification curves are less than 20, showing that the system of the present invention can sensitively detect all randomly selected clinical strains of Klebsiella pneumoniae, proving that the detection system of the present invention It has a high prevalence against Klebsiella pneumoniae.

[0071] The results show that the characteristic sequence of the present invention obtains positive signals for randomly selected clinical strains of Klebsiella pneumoniae, which proves that the characteristic sequence of the present invention exists in most clinical strain...

Embodiment 3

[0073] Escherichia coli (E.coli), Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter baumannii (A.baumanni), Salmonella (S.enterica) and Staphylococcus aureus (S. .aureus) clinical isolates, 19 strains were selected at random, and were detected by the detection method of the present invention. The qPCR reaction system and reaction procedure of detection are the same as in Example 1, and the detection results are shown in Image 6 .

[0074] Depend on Image 6 The test results showed that all clinical strains had no amplification signal when Ct=20 was used as the cutoff value. It proves that the characteristic sequence of the present invention does not exist in most clinical strains such as E. Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, etc. can be distinguished with high specificity and low false positive rate.

[0075] Based on the results of Examples 1, 2, and 3, it can be concluded that based on the characteristic sequence of the present invention, Kle...

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Abstract

The invention belongs to the technical field of klebsiella pneumoniae detection, and particularly relates to a klebsiella pneumoniae characteristic sequence and a qPCR rapid detection system and method thereof, wherein the klebsiella pneumoniae characteristic sequence is shown as SEQ ID NO: 1, and the primer group sequence for amplifying the klebsiella pneumoniae characteristic sequence is shown as SEQ ID NO: 2 and SEQ ID NO: 3. According to the invention, the characteristic sequence can be used for preparing a klebsiella pneumoniae nucleic acid detection kit; according to the qPCR detection method for klebsiella pneumoniae, the sensitivity reaches the picogram level, klebsiella pneumoniae can be sensitively distinguished from other common respiratory tract bacteria, and klebsiella oxytoca which is the related bacteria of klebsiella pneumoniae can also be distinguished; and the klebsiella pneumoniae characteristic sequence has the characteristics of high sensitivity, strong specificity, wide coverage and the like, can effectively reduce the false negative rate and false positive rate of clinical detection of klebsiella pneumoniae, and has important significance for improving the clinical treatment effect of klebsiella pneumoniae.

Description

technical field [0001] The invention belongs to the technical field of detection of Klebsiella pneumoniae, and in particular relates to a characteristic sequence of Klebsiella pneumoniae and a qPCR rapid detection system and method thereof. Background technique [0002] Surveillance data released by the China Antimicrobial Resistance Surveillance Network (CHINET) in 2019 showed that Klebsiella pneumoniae is the most common pathogenic bacteria for respiratory tract infections. For details, see figure 1 . In fact, in recent years, the detection rate of Klebsiella pneumoniae in clinical infections has been increasing year by year. Before 2017, the most common pathogen of respiratory tract infection was Acinetobacter baumannii. [0003] At the same time, the clinical isolates of Klebsiella pneumoniae in my country have become very resistant to a variety of commonly used antibiotics. The monitoring results of CHINET in 2019 showed that more than 50% of clinical isolates of Klebs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/10C12N15/11C12R1/22
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 高帆宋韡杨鹏
Owner SHANXI UNIV