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Constant-temperature amplification detection method for klebsiella acidophilus and special primer and kit thereof

A technology for Klebsiella oxytoca and Klebsiella spp. is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. , to achieve the effect of simple identification of results and simple operation

Inactive Publication Date: 2019-05-14
AIR FORCE MEDICAL CENT PLA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional detection methods of Klebsiella oxytoca, including lactic acid bacteria degradation analysis test, melezitose utilization test, API identification method and VITEK2 automatic bacterial identification method, etc., are usually based on phenotypic detection, so it is difficult to identify Klebsiella oxytoca. Differentiation of bacilli from other Klebsiella genus bacteria such as Klebsiella pneumoniae
Currently commonly used molecular genotyping methods, such as pulsed field gel electrophoresis, random amplified polymorphic DNA analysis, and amplified fragment length polymorphism analysis, have high requirements for experimental equipment and operators, and are not suitable for grassroots Medical institution promotion

Method used

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  • Constant-temperature amplification detection method for klebsiella acidophilus and special primer and kit thereof
  • Constant-temperature amplification detection method for klebsiella acidophilus and special primer and kit thereof
  • Constant-temperature amplification detection method for klebsiella acidophilus and special primer and kit thereof

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Comparison scheme
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Embodiment 1

[0078] Example 1. Primer Design for LAMP Detection of Klebsiella oxytoca

[0079] 1. Primer design for LAMP detection of Klebsiella oxytoca

[0080] The Klebsiella oxytoca sequence (GenBank number: AY065648.1) was retrieved from the American gene database GenBank, and five sets of primer combinations were designed for LAMP detection of Klebsiella oxytoca with the software Primer Explorer Ver.5, respectively. For KO-1, KO-5, KO-9, KO-13 and KO-16, the primer sequences are as follows in Table 1:

[0081] Table 1 is the alternative primers for LAMP detection

[0082]

[0083]

[0084] Two, the establishment of the LAMP detection method of Klebsiella oxytocin of the present invention and the screening of optimal primer

[0085] Use the 5 sets of primers obtained above for LAMP detection of Klebsiella oxytoca to perform LAMP detection on Klebsiella oxytoca standard strain K. Next, add different primer combinations KO-1, KO-5, KO-9, KO-13, KO-16 in the reaction system to de...

Embodiment 2

[0098] Example 2, the specificity and sensitivity detection of the LAMP detection method of Klebsiella oxytoca in the present invention

[0099] One, the specificity detection of the LAMP detection method of Klebsiella oxytocia of the present invention

[0100] 1. LAMP reaction

[0101] Genomic DNA was extracted from the following strains:

[0102] 1,K.oxytoca ATCC 700324;2,Klebsiella pneumoniae ATCC 2146;3,Klebsiella pneumoniae ATCC 7105;4,Klebsiella rhinoscleromatis CMCC46111;5,Haemophilus influenza ATCC49247;6,Salmonella typhi 9275;7,Streptococcuspneumoniae 112-07;8,Escherichia coli 44825;9,Pseudomonas aeruginosa ATCC15442;10,Shigella flexneri4536;11,Legionella pneumophila 9135;12,Proteusvulgaris CMCC49027;13,Mycobacterium tuberculosis 005;14,Acinetobacterbaumannii 12101;15,Stenotrophomonas maltophilia K279a;16,negative control(double-distilled water).

[0103] 1) Real-time turbidity LAMP method

[0104] Real-time turbidity LAMP method reaction system: the total volume is...

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Abstract

The invention discloses a constant-temperature amplification detection method of klebsiella acidophilus and a special primer and a kit thereof. The constant-temperature amplification detection methodof klebsiella acidophilus and the special primer and the kit thereof provides a complete set of reaction primers for detecting loop-mediated isothermal amplification of klebsiella acidophilus, the setof reactive primer comprises the primer F3, the primer B3, the primer FIP, the primer BIP, the primer LoopF and the primer LoopB. The nucleic acid sequences corresponding to the primer F3, the primerB3, the primer FIP, the primer BIP, the primer LoopF and the primer LoopB are respectively sequence 1 to sequence 6 in the sequence table. The primer of the invention can rapidly, conveniently, efficiently, highly specifically and highly sensitively detect klebsiella acidophilus under isothermal conditions, does not need complex instruments, provides a new technical platform for the detection ofthe klebsiella acidophilus, can be used for screening and detecting klebsiella acidophilus in basic medical care units and disease prevention and control centers, has wide market prospect and great economic and social benefits, and is suitable for wide popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a constant-temperature amplification detection method for Klebsiella oxytoca, as well as special primers and a kit. Background technique [0002] Klebsiella is an important pathogen of community infection and hospital infection, especially in immunocompromised patients and ICU critically ill patients, it can cause severe lung infection and bloodstream infection, with high morbidity and mortality. The genus Klebsiella mainly includes 5 species of Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella ornithinolyticus, Klebsiella phytogenes and Klebsiella terreus, of which Klebsiella pneumoniae Bacillus and Klebsiella oxytoca are important pathogens that cause lung and bloodstream infections in humans. In addition to causing lung and bloodstream infections, Klebsiella oxytoca is an important pathogen of food poisoning and antibiotic-associated hemorrhagic colitis. Studies ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
Inventor 赵进董德荣刘运喜张波刘一董志伟
Owner AIR FORCE MEDICAL CENT PLA