Primer and probe, kit and method for detecting Proteus mirabilis

A technology of Proteus mirabilis and kits, applied in the field of microbial detection, can solve the problems of low efficiency, easy to produce false positives, long cycle, etc., and achieve the effects of convenient use, good specificity, and low cost

Active Publication Date: 2020-02-04
SHENZHEN CENTER FOR DISEASE CONTROL AND PREVENTION (SHENZHEN HEALTH INSPECTION CENTER SHENZHEN INSTITUTE OF PREVENTIVE MEDICINE)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art, and provide a primer, a probe, a kit, and a method for quickly and accurately detecting Proteus mirabilis, so as to solve the problem that the current detection method of Proteus mirabilis has a long period, low efficiency, and is prone to false positives. And other issues

Method used

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  • Primer and probe, kit and method for detecting Proteus mirabilis
  • Primer and probe, kit and method for detecting Proteus mirabilis
  • Primer and probe, kit and method for detecting Proteus mirabilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1 Proteus mirabilis detection kit

[0062] 1. Design primers and probes for target sequence one obtained by whole gene sequencing, target sequence two, target sequence three and target sequence four, synthesize the nucleotide sequences and Homo-tag sequences of its primers and probes (as shown in Table 1 DABCYL is connected to the 3' end of each probe sequence, and the fluorophore corresponding to the 5' end of each probe sequence is: SEQ ID NO: 3 corresponds to HEX, SEQ ID NO: 6 corresponds to ROX, and SEQ ID NO: 6 corresponds to ROX. ID NO: 9 corresponds to CY5 and SEQ ID NO: 12 corresponds to FAM.

[0063] Each of the above primers and probes was prepared as a storage solution with a concentration of 0.2 μM, and the Homo-tag sequence was prepared as a storage solution with a concentration of 0.6 μM.

[0064] 2. Separately prepare PCR buffer, MgCl 2 solution, dNTP solution, rTaq enzyme solution; among them, MgCl 2 The concentration is 3mM, the dNTP conce...

Embodiment 2

[0066] The detection and analysis of embodiment 2 Proteus mirabilis

[0067] 1. Sample nucleic acid extraction: Take about 0.2g or 200μl of stool samples from patients with diarrhea caused by clinical Proteus mirabilis and healthy people respectively, add them to 1.5ml EP tubes, add the sample processing solution (PBS buffer or normal saline), Shock 3 times, 10 seconds each time. Let it stand for 10 minutes, then centrifuge at 8000rpm for 5 minutes, draw the supernatant and directly boil it for nucleic acid extraction to obtain 10-100ng / μl nucleic acid template.

[0068] 2. Fluorescent quantitative PCR reaction:

[0069] Take the detection kit prepared in Example 1, add 5.0 μl of the nucleic acid template obtained in the above step 1 to each PCR reaction tube, and the components of the 25 μl reaction system in each PCR reaction tube are as follows:

[0070]

[0071]

[0072] Then, send the PCR reaction eight tubes into a fluorescent PCR instrument (staragene-3005) for ...

Embodiment 3

[0079] Embodiment 3 specificity analysis

[0080] The applicant simultaneously obtained 194 strains involving bacteria such as Enterobacter, cocci, and Vibrio, and used the detection kit of Example 1 to carry out specific analysis experiments, and used the positive strain of Proteus mirabilis as a control. The situation of the detected strains is shown in Table 3. The test results were all negative. The results of this example show that: the primers and probes in the kit provided in Example 1 of the present invention have good specificity, and no non-specific amplification occurs, thereby avoiding false positives caused by non-specificity.

[0081] table 3

[0082]

[0083]

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Abstract

The invention belongs to the technical field of microbiological detection, and in particular, relates to primers, probes, a kit and a method for detecting proteus mirabilis. The invention discloses a set of primers and probes for detecting the proteus mirabilis, wherein the set of primers and probes comprise primers and probes for detecting four target sequences. The kit for detecting the proteus mirabilis comprises the primers and probes for detecting the proteus mirabilis. The primers and probes provided by the invention can accurately detect the proteus mirabilis, have good specificity and high sensitivity, and avoid generation of false positives; and the kit is low in cost, easy to use, and suitable for rapid screening for the proteus mirabilis.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a primer, a probe, a kit and a method for detecting Proteus mirabilis. Background technique [0002] Proteus mirabilis (PM) belongs to the genus Proteus and is a resident bacterium in the intestinal tract of humans and animals. It is an opportunistic pathogenic bacterium that can cause intestinal infection under certain conditions. Certain serotype strains are highly pathogenic and can cause diarrhea through diet. Proteus mirabilis is a common pathogen of bacterial diarrhea. PM infection is mainly seen in infants and young children. In recent years, its detection rate in adult diarrhea patients has increased, and it has become the main pathogenic bacteria causing diarrhea in some areas, and drug resistance to commonly used drugs has gradually emerged, so it has received extensive attention. . Most of the clinical symptoms caused by PM are watery diar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/10C12N15/11C12R1/37
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2531/113C12Q2545/113C12Q2537/143C12Q2563/107
Inventor 石晓路扈庆华袁建辉姜伊祥林一曼邱亚群李迎慧江敏陈琼城白芳
Owner SHENZHEN CENTER FOR DISEASE CONTROL AND PREVENTION (SHENZHEN HEALTH INSPECTION CENTER SHENZHEN INSTITUTE OF PREVENTIVE MEDICINE)
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