Proteusbacillus vulgaris multi-serotype strain specific primer and multiple polymerase chain reaction (PCR) detection method

A proteus, specific technology, applied in the field of multiplex PCR rapid detection, can solve the problems of time-consuming, missed detection, antiserum limitation, etc.

Active Publication Date: 2014-11-26
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional method of detecting pathogenic microorganisms is to identify the bacteria at the biochemical and serological levels after culturing, which requires at least 48 hours of bacterial culture and single colony analysis, and requires the use of multiple antibiotics for a large number of single colonies in each sample. Serum agglutination reaction takes a long time, and there is a possibility of missed detection
At the same time, there is another defect in the serological method, which is limited by antiserum. No company or unit in the world can produce antiserum for all bacteria.

Method used

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  • Proteusbacillus vulgaris multi-serotype strain specific primer and multiple polymerase chain reaction (PCR) detection method
  • Proteusbacillus vulgaris multi-serotype strain specific primer and multiple polymerase chain reaction (PCR) detection method
  • Proteusbacillus vulgaris multi-serotype strain specific primer and multiple polymerase chain reaction (PCR) detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 : Genome Extraction

[0127] Proteus was cultured in a small shaking tube equipped with 2YT, and the cells were collected after overnight culture at 37°C and 180rpm in a shaker, and the genome was extracted by traditional phenol and chloroform extraction method. Specific steps are as follows:

[0128] (1) Collect 1ml of the bacterial liquid into a 1.5ml centrifuge tube, centrifuge at 12000rpm for 10min, discard the supernatant to collect the bacterial cells.

[0129] (2) Add 500 μl tris-Hcl to the centrifuge tube to resuspend the cells, and centrifuge at 12,000 rpm for 5 minutes to remove the supernatant.

[0130] (3) Repeat step (2)

[0131] (4) Add 250ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA to the cells to resuspend the cells, add 10ul 10mg / ml lysozyme and incubate at 37°C for 20 minutes. For Gram-positive bacteria overnight.

[0132] (5) Add 3ul 20mg / ml proteinase K and 15ul 10% SDS to the mixture, incubate at 50°C for 2 hours. If the liquid does...

Embodiment 2

[0138] Embodiment 2: the design of primer

[0139] Using the Proteus sequence tested by the laboratory, for serotypes 1, 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 17 Type, Type 18, Type 19, Type 21, Type 23, Type 24, Type 25, Type 26, Type 27, Type 28, Type 29, Type 30, Type 31, Type 32, Type 33, Type 34, Type 36, Type 37, Type 38, Type 39, Type 40, Type 41, Type 44, Type 45, Type 47, Type 48, Type 50, Type 52, Type 53, Type 54, Type 55, Type 56, Type 57, Type 58 , Type 59, and Type 60's wzy Primers were designed for the specific regions of the genes, and the primer sequences are shown in Table 1 below:

[0140] Table 1 Sequences of specific primers for 48 serotypes of Proteus

[0141]

[0142]

[0143]

[0144]

[0145]

Embodiment 3

[0146] Example 3 : Screening of specific primers

[0147] Primers were designed for specific regions of the wzt gene of 48 Proteus serotypes, and the primer sequences are shown in Table 1 (SEQ ID NO:1-SEQ ID NO:96). One standard strain of each of the 48 Proteus serotypes, one strain of each of the 10 standard strains of other Proteus serotypes, and six closely related bacteria were collected to verify the specificity of the primers. The strain numbers and sources are shown in Table 2 below.

[0148] Table 2 Standard strains for testing

[0149]

[0150]

[0151]

[0152]

[0153] The PCR system is 10uM primers are added according to the instructions in Table 3, MgCl 2 3μl, 10×buffer 3μl, 10mM dNTP 2.4μl, 5 U / μl Taq polymerase 0.3μl and 2μl sample template to be tested into a 0.2ml thin-walled PCR tube, and finally use ddH 2 O to make up to 30 μl. All primers obtained positive results in the 48 Proteus templates within the detection range, and did not get an...

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Abstract

The invention relates to a specific polymerase chain reaction (PCR) primer for detecting proteusbacillus vulgaris multi-serotype pathogenic bacteria, a multiple PCR rapid detection method and application of the method. The multiple PCR detection system comprises a 10*PCR reaction solution, MgCl2, dNTP, a primer and DNA polymerase, wherein the sequence of the primer comprises the following multiple sequences: (1) wzy specific nucleotide sequences of 1-4 type to type 60 of proteusbacillus vulgaris serotype; and (2) a complementary DNA sequence of the DNA sequence selected from (1). According to the wzy specific nucleotide for common proteusbacillus vulgaris serotype and the multiple PCR detection method comprising the nucleotide, a multiple PCR system is easy and convenient in preparation method, short in detection period and high in speed, accuracy and operability, and the detection cost is reduced.

Description

technical field [0001] The present invention relates to a multiplex PCR rapid detection method and its application, in particular to a PCR technique that can be used to simultaneously detect 48 bacterial strains from Proteus serotypes 1-4 to type 60 and the sequence of the PCR primers used . Background technique [0002] Proteus ( Proteus ) belongs to Gram-negative bacteria, widely exists in water, soil decayed organic matter, and human and animal intestines, plays an important role in decomposing organic matter and animal protein, and has Activity of proteolytic enzymes. Proteus is an opportunistic pathogen, mostly secondary infection, such as chronic otitis media, trauma infection, etc., and can also cause cystitis, infant diarrhea, food poisoning, etc. [0003] The current latest classification defines the genus Proteus vulgaris including Proteus vulgaris ( P. vulgaris ), Proteus mirabilis ( P. mirabilis ), Proteus pennei ( P. penneri ), Proteus mucogenes ( P. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 王磊王荃彭志勇冯露
Owner NANKAI UNIV
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