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Pathogen detection method

A detection method and technology for pathogenic bacteria, which are used in the determination/inspection of microorganisms, biochemical equipment and methods, and resistance to vector-borne diseases, etc. problem, to achieve the effect of rapid identification

Inactive Publication Date: 2010-07-07
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a detection method for pathogenic bacteria in view of the defects of the existing bacteria detection operation, such as complicated operation, long detection period, difficulty in obtaining specific monoclonal antibodies and high price in bacteria-specific detection, etc. The detection method utilizes genetically modified phages to identify pathogenic bacteria simply, rapidly, sensitively and specifically

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Preparation of multivalent phage M13KE-TC

[0025] Preparation of tetracysteine ​​TC fragment:

[0026] Two TC DNA complementary sequences with Acc65 I and Eag I cohesive ends were designed and synthesized in Sansheng.

[0027] P1: 5'-GTACCTTTTCTATTCTCACTCTTTTCCTGAACTGTTGTCCCGGCTGCTGCATGGAGCCTTC-3' (denoted as SEQ ID NO.1);

[0028] P2: 5'-GGCCGAAGGCTCCATGCAGCAGCCGGGACAACAGTTCAGGAAAGAGTGAGAATAGAAAG-3' (denoted as SEQ ID NO. 2).

[0029] Take P1 and P2 respectively and mix them in Taq Buffer at a final concentration of 10M, and perform annealing reaction in a PCR instrument. The setting program is as follows: 95°C for 2min; continuously lower to 25°C for about 60min; store at 4°C.

[0030] The 7222bp multivalent phage M13KE DNA was recovered by double digestion with Eag I and Acc65 I. After the double digestion fragment was ligated with the annealed TC DNA fragment with sticky ends of Eag I and Acc65 I, it was transfected into E. coli TG 1. In the state, pla...

Embodiment 2

[0051] Example 2 Preparation of monovalent phage M13KO7-TC

[0052] 1. Construction and identification of recombinant phagemid pCANTAB 5E-TC

[0053]Two TC DNA complementary sequences with Not I and Sfi I cohesive ends were designed and synthesized in Sansheng.

[0054] P3: 5`-CGGCCATTCCTGAACTGTTGTCCCGGCTGCTGCATGGAGCCTGC-3` (denoted as SEQID NO.3);

[0055] P4: 5`-GGCCGCAGGCTCCATGCAGCAGCCGGGACAACAGTTCAGGAATGGCCGGCT-3`

[0056] (Denoted as SEQ ID NO.4). Take P3 and P4 respectively and mix them in Taq Buffer at a final concentration of 10 M, and perform annealing reaction in a PCR instrument. The setting program is as follows: 95°C for 2 minutes; continuously lower to 25°C for about 60 minutes; store at 4°C.

[0057] The 5.3kb phagemid DNA pCANTAB 5E was digested with Not I and Sfi I, recovered, and the double digested fragment was ligated with the annealed TC DNA fragment with Not I and Sfi I sticky ends to construct a recombinant phagemid. Cloning and sequencing were carri...

Embodiment 3 4

[0083] Example 3 Infection and double arsenic dye staining of specific pathogenic bacteria by tetracysteine ​​recombinant bacteriophage

[0084] Pick a single colony of E.coli TG1 to be tested and culture it overnight in 2mL 2×YT, 37°C, 250rpm, take 100L of the overnight cultured E.coliTG1 bacteria solution and add it to 10mL 2×YT-G, shake to OD at 37°C, 250rpm =0.6, take 2 mL of the corresponding bacterial solution in a culture tube, add recombinant phage and prophage respectively, infect at 37° C., 250 rpm for 1 hour. Take 80uL of each infection solution, wash twice with 100L HBSS, finally add HBSS, and add the double arsenic dye FlAsH-EDT with a final concentration of 5μM 2 , incubated for 30 min, centrifuged to discard the supernatant, added detergent BAL, incubated for 15 min, centrifuged to discard the supernatant, repeated washing once, added HBSS 80uL to resuspend. In this way, the bacterial samples infected with the prophage are the negative control group; the bacter...

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Abstract

The invention discloses a pathogen detection method, which relates to a pathogenic microorganism detection method. The invention provides the pathogen detection method, which utilizes genetically engineered phage to identify the pathogen simply, rapidly, sensitively and specifically. The pathogen detection method comprises the following steps: designing and synthetizing a nucleotide sequence with a cohesive terminus, wherein a coding of the nucleotide sequence is provided with 4-cysteine polypeptide; operably connecting the nucleotide sequence of which the coding is provided with the 4-cysteine polypeptide in an expressed regulatory sequence of the capsid protein of phage to form 4-cysteine recombinant phage; using the recombinant phage to infect host cells to make the recombinant phage reproduce in the host cells reproduce to generate recombinant cells with expressed 4-cysteine recombinant phage; and adding bi-arsine dye in the recombinant cells, removing nonspecific binding by washing and using a flow cytometry or a fluorescence microscope to carry out specific detection because the pathogen specifically identified and infected by the recombinant phage can emit specific fluorescence when being irradiated by exciting light.

Description

technical field [0001] The invention relates to a method for detecting pathogenic microorganisms, in particular to a method and application for rapid differential diagnosis of pathogenic bacteria by a double arsenic dye-tetracysteine ​​recombinant bacteriophage. Background technique [0002] Rapid, sensitive, and specific detection and analysis of pathogenic bacteria is of great significance to food safety, environmental monitoring, disease diagnosis and treatment, and prevention of bioterrorism attacks. The traditional detection methods of pathogenic microorganisms require separation, cultivation and a series of biochemical reactions of bacteria. The operation is complex, the detection period is long, and it cannot be applied to pathogenic bacteria that are difficult to cultivate, which is far from meeting the actual needs ([1] Iqbal, S.S., et al ., A review of molecular recognition technologies for detection of biological threat agents. Biosens Bioelectron, 2000.15(11-12):...

Claims

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Application Information

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IPC IPC(8): C12Q1/10C12Q1/04G01N21/64
CPCY02A50/30
Inventor 吴丽娜黄婷婷潘建波颜晓梅
Owner XIAMEN UNIV
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