System for detecting and distinguishing proteus mirabilis, proteus vulgaris and proteus pennerei and method thereof

A technology of Proteus mirabilis and Proteus vulgaris, applied in the direction of microorganism-based methods, biochemical equipment and methods, and measurement/inspection of microorganisms, can solve the problems of inability to distinguish and quantify Proteus mirabilis, Proteus panethii, time-consuming, poor specificity

Active Publication Date: 2020-04-03
CHONGQING ACAD OF ANIMAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no method that can quickly detect Proteus infection and accurately distinguish the three Proteus species within the genus Proteus.
Among the existing methods, the biochemical identification method has the disadvantages of cumbersome operation, long time-consuming, and poor specificity; while the common PCR and fluorescence quantitative PCR methods that have been established at present can only distinguish Proteus mirabilis from Proteus, and cannot Differentiation and quantification of P. vulgaris and P. penneri
It can be seen that there are relatively large defects in the existing Proteus detection technology.

Method used

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  • System for detecting and distinguishing proteus mirabilis, proteus vulgaris and proteus pennerei and method thereof
  • System for detecting and distinguishing proteus mirabilis, proteus vulgaris and proteus pennerei and method thereof
  • System for detecting and distinguishing proteus mirabilis, proteus vulgaris and proteus pennerei and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Multiplex TaqMan Real-Time PCR primer set for detection of 3 species of Proteus.

[0040]The present invention analyzes the sequences of all Proteus mirabilis, Proteus vulgaris, Proteus pannei and Proteus huisii published on GeneBank when designing primers, and compares them with Providencia and Morganella which belong to Enterobacteriaceae Compared the nucleotide sequences of Proteus, etc., and used the method of constructing a phylogenetic tree to screen and detect the detection target genes, and finally found that the tur gene can distinguish Proteus from other genera, and can also distinguish Proteus mirabilis , Proteus vulgaris, Proteus panethii and Proteus huisii. The ureR gene can effectively distinguish Proteus mirabilis from other Proteus. In order to better optimize the detection primers and probes, the tur gene was selected as the target gene for detecting Proteus vulgaris and Proteus panethii; The nucleotide sequences of the designed primers and ...

Embodiment 2

[0053] Example 2 Multiplex TaqMan Real-Time PCR kit for detecting 3 kinds of Proteus.

[0054] The invention provides a multiplex TaqMan Real-Time PCR kit for detecting Proteus and simultaneously distinguishing three kinds of Proteus. The kit contains the pre-reaction system composed of the primer set and probe set, qPCR Mix and ddH2O in Example 1, and the specific content is as follows:

[0055] The concentration of each primer (SEQ ID NO:1-SEQ ID NO:6) in the primer set is 5 μ mol / L; The concentration of each probe (SEQ ID NO:7-SEQ ID NO:9) in the probe set is 5 μmol / L; qPCR Mix is ​​produced by Takara Company, containing Taq enzyme, PCR buffer, dNTP, MgCl2; ddH2O is deionized water without DNase and RNase.

[0056] The optimized 40μL reaction system includes the pre-reaction system (qPCR Mix 20.0uL, primer set 3.0μL, probe set 3.0μL, H2O 9.0uL) and template DNA (10-100ng) 5uL, mix the two and centrifuge Perform PCR reaction.

Embodiment 3

[0057] Example 3 Amplification procedure of multiplex TaqMan Real-Time PCR for detection of 3 kinds of Proteus.

[0058] Place the 0.1mL PCR eight-tube or 96-well plate with the reaction system in the fluorescent quantitative PCR instrument with three channels, select three fluorescent channels: FAM, HEX and Red610; set the amplification program: the first step , 94°C hot start and pre-denaturation for 5min; second step, 94°C denaturation for 10s; third step, 60°C annealing for 30s, and detection of fluorescence in the third step; the second step and the third step totaled 40 cycles.

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Abstract

The method is suitable for the technical field of bacteria detection, the invention discloses a triple TaqMan Real-Time PCR detection method for simultaneously distinguishing proteus mirabilis(P. mirabilis), proteus vulgaris (P.vulgaris) and proteus penesei (P.pennerei), and a kit. The primers and nucleotide sequences of proteusbacillus vulgaris are predicted through computer software, and three pairs (six) of primers capable of being used for simultaneously detecting three proteusbacillus vulgaris and nucleotide sequences of three probes are disclosed. The method has the characteristics of strong specificity, high sensitivity and accurate and reliable result. According to the method, the proteusbacillus vulgaris can be identified as a specific species while the proteusbacillus vulgaris isdetected, the defect that only proteus mirabilis can be distinguished in existing proteusbacillus vulgaris detection is overcome, the proteusbacillus vulgaris detection process is greatly simplified,the workload of operators is reduced, the detection period is shortened, and a powerful technical support can be provided for detecting the proteusbacillus vulgaris in the industries of medical treatment, medical detection and the like.

Description

technical field [0001] The invention belongs to the technical field of bacteria detection, and is a triple TaqMan Real-Time PCR system for detecting Proteus and simultaneously distinguishing Proteus mirabilis, Proteus common and Proteus panethii Background technique [0002] Proteus is a Gram-negative bacterium belonging to the family Enterobacteriaceae, which exists widely in nature and is an opportunistic pathogen in the intestinal tract of humans and animals. The genus Proteus includes four species: P. mirabilis, P. vulgaris, P. penneri and P. hauseri. Among them, P. mirabilis, P. vulgaris, and P. penneri are considered pathogenic and persist in various combinations in the gut of healthy humans and animals for a long time, but the abundance is very low (<0.05%); Bacilli are commonly found in water and are not considered to cause disease. P.mirabilis, P.vulgaris and P.penneri can cause various diseases such as food poisoning, urinary tract infection, wound and burn in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/37
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2561/101C12Q2561/113
Inventor 杨睿付利芝王孝友徐登峰曹政余远迪张素辉杨柳牟豪许国洋
Owner CHONGQING ACAD OF ANIMAL SCI
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