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Kit for detecting clinically common pathogenic bacteria by adopting RNA isothermal amplification melting curve method and applications of kit

A technology of constant temperature amplification and melting curve, which is applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of false negative detection cost, long detection cycle, low sensitivity, etc., and achieve high specificity , high specificity and high sensitivity

Active Publication Date: 2017-06-20
温州迪安医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a test kit for detecting common clinical pathogenic bacteria by RNA constant temperature amplification melting curve method and its application, so as to solve the problem of low sensitivity of the detection method of common clinical pathogenic bacteria in the prior art and the detection cycle Long time, easy contamination, false positive or false negative, and high detection cost

Method used

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  • Kit for detecting clinically common pathogenic bacteria by adopting RNA isothermal amplification melting curve method and applications of kit
  • Kit for detecting clinically common pathogenic bacteria by adopting RNA isothermal amplification melting curve method and applications of kit
  • Kit for detecting clinically common pathogenic bacteria by adopting RNA isothermal amplification melting curve method and applications of kit

Examples

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Effect test

Embodiment 1

[0044] Example 1: Design of RNA Constant Temperature Amplification Primers and Melting Curve Detection Molecular Beacon Probes

[0045] Due to the conservation and universality of the 16S rRNA gene sequence, the use of 16S rRNA as a molecular indicator has gradually become a powerful tool for microbial detection and classification. The design idea of ​​molecular beacon probes is to comprehensively consider the specificity and generality of the detection of common pathogenic bacteria, and the universal principles that should be followed in the design of primers and probes (such as Tm value, 3' terminal free energy, GC content, Avoid the appearance of internal structure and dimer formation, etc.), and the impact of the reverse primer plus the T7 phage promoter sequence, etc. After the designed primers and probes are synthesized, they are screened and verified by RT-PCR experiments of 20 common pathogenic bacteria and RNA constant temperature amplification melting curve reaction ...

Embodiment 2

[0058] Example 2: Establishment and optimization of detection system for common clinical pathogenic bacteria RNA constant temperature amplification melting curve method

[0059] Conditions were optimized for some important factors affecting the detection system of the RNA constant temperature amplification melting curve method.

[0060] 1. Method

[0061] (1)Mg 2+ Concentration: Prepare 2× constant temperature amplification reaction solution according to the reaction system in Table 1, fix other parameters, and adjust Mg 2+ The final concentration was 4mM, 8mM, 12mM, 16mM, 20mM, 24mM, and the total RNA of Enterococcus faecium extracted was used as template for constant temperature amplification and isothermal amplification. After the experiment, compare the different Mg 2+ Effect of concentration on amplification efficiency and melting curve.

[0062] (2)K + Concentration: prepare 2× constant temperature amplification reaction solution according to the reaction system in ...

Embodiment 3

[0067] Embodiment 3: Composition and detection method of clinical common pathogenic bacteria (RNA constant temperature amplification melting curve method) kit

[0068] 1. Composition of the kit (stored at -20°C)

[0069] (1) 2× constant temperature amplification reaction solution: its components are: 80mM Tris-HCl (pH8.0), 140mM KCl, 24mMMgCl 2 , 10mM DTT, 2mM dNTP, 4mM NTP, Q-solution, 0.4μM forward primer F1(F2), 0.4μM reverse primer R1(R2), 0.05μM molecular beacon probe P1(P2), 0.1μM molecular signal Marking probe P3; where the forward primer F1 is the nucleotide sequence shown in SEQ ID NO: 1, the reverse primer R1 is the nucleotide sequence shown in SEQ ID NO: 2, the forward Primer F2 is the nucleotide sequence shown in SEQ ID NO: 3, the reverse primer R2 is the nucleotide sequence shown in SEQ ID NO: 4, and the molecular beacon probe P1 is the nucleotide sequence shown in SEQ ID NO: 4. The nucleotide sequence shown in SEQ ID NO: 5, the molecular beacon probe P2 is the ...

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Abstract

The invention discloses a kit for detecting clinically common pathogenic bacteria by adopting an RNA isothermal amplification melting curve method and applications of the kit, belonging to the technical field of biological detection. The kit can detect the 16S rRNA of the following 16 clinically common pathogenic bacteria: staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, proteus mirabilis, enterobacter aerogenes, pseudomonas fluorescens, acinetobacter baumannii, salmonella typhimurium, enterobacter cloacae, enterococcus faecium, enterococcus faecalis, bacillus proteus vulgaris, staphylococcus epidermidis, onion klebsiella and stenotrophomonas maltophilia. The kit has the features of high specificity, high sensitivity, low pollution, and detection accuracy and rapidness, plays an important role in clinical rapid identification and detection analysis of microbes, and has the wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a kit and a detection method for detecting common clinical pathogenic bacteria by RNA constant temperature amplification melting curve method. It is suitable for the identification of common clinical pathogenic bacteria, and can provide guidance for rational antibacterial drug treatment for patients with clinical bacterial infections. Background technique [0002] Bacterial infection is a common clinical infectious disease with high morbidity and mortality. If it is not diagnosed and treated early, it will seriously endanger the lives of patients. Bacteria isolated directly from clinical samples indicate a serious infection requiring immediate antimicrobial therapy. Different pathogenic bacteria have different sensitivities to drug types and concentrations, and the key to successful treatment lies in the early application of appropriate and sufficient drugs. However,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2600/158C12Q2600/178C12Q2527/101C12Q2527/107C12Q2563/107Y02A50/30
Inventor 任绪义罗英
Owner 温州迪安医学检验所有限公司
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