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Universal primer PCR method for immunocapture to detect bacteria

A technology of universal primers and bacteria, applied in biochemical equipment and methods, measuring devices, and microbial determination/inspection, etc., can solve the problems of difficult to achieve rapid specific detection, complicated operation steps, etc., and achieve clear PCR product bands, Improved sensitivity and high repeatability

Inactive Publication Date: 2002-08-28
XIAMEN UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

In recent years, PCR technology has been applied to the detection of pathogenic bacteria. These detection technologies can be divided into two categories, namely PCR with specific primers and PCR with non-specific primers. The former often requires different primers depending on the target bacteria, while different primers The sequence may require different amplification conditions. Therefore, when the bacteria to be tested are unknown, multiple PCRs are required to obtain the results. Therefore, it is difficult to achieve the purpose of rapid and specific detection; the latter is generally synthesized based on the conservation of the bacterial 16S rRNA gene Universal primers, the amplified products often need to cooperate with RFLP (restriction fragment polymorphism analysis), SSCP (single-strand conformation polymorphism analysis) or sequence analysis to be confirmed, so the operation steps are more cumbersome

Method used

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  • Universal primer PCR method for immunocapture to detect bacteria
  • Universal primer PCR method for immunocapture to detect bacteria
  • Universal primer PCR method for immunocapture to detect bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Experimental strains: Shigella dysenteriae type I, Shigella sonnei, Shigella flexneri 1a, 2a, 2b, 3a, 4, 5, Y varieties, abalone Shigella boydii 1 . Among them, Shigella dysenteriae type I, Shigella sonneri, Shigella flexneri 4, Shigella baumannii 16 were purchased from the Jiangxi Provincial Health and Epidemic Prevention Station, Shigella flexneri 1a, 2a, 2b, 3a, 5, The Y variant was purchased from the Health and Epidemic Prevention Station of Fujian Province.

[0016] Universal primer: Synthesized by Sangon Company, selected from the conserved region of bacterial 16S rRNA gene, identified as pure by electrophoresis. The primer sequences are as follows: forward primer 5'-AAACTCAAAGGAATTGACGG-3'reverse primer 5'-GACGGGCGGTGTGTACAA-3'.

[0017] Diagnostic serum: The diagnostic serum of the above-mentioned Shigella bacteria is the product of the Lanzhou Institute of Biological Products, Ministry of Health, and the antibodies are purified by the 4-step ammonium sulfate ...

Embodiment 2

[0030] Neutralization experiment: set up a neutralization group and a control group at the same time to carry out the neutralization experiment. The materials and iUPPCR operation steps are the same as those in Example 1. Dilute Shigella dysenteriae type I, Shigella sonei, Shigella flexneri 4, and Shigella baumannii 16 into 20 μL of bacterial liquid containing 50 CFU of pathogenic bacteria . In the neutralization group, 20 μL of bacterial solution was mixed with an equal volume of 50 μg / mL, 100 μg / mL, and 200 μg / mL of the corresponding antibody, incubated at 37°C for 1 hour, and then added to the reaction plate; for the control group, 20 μL of bacterial solution and 20 μL of sterile water were added. , and the rest of the steps remain unchanged. The neutralization results are shown in Table 1. There was no PCR amplification product in the neutralization group of the three groups, while the corresponding control group had amplification products.

[0031] Table 1: Neutralizati...

Embodiment 3

[0034] Embodiment 3: iUPPCR cross experiment

[0035] 1) Crossover experiments were carried out with purified antibodies from species-specific monovalent diagnostic serum: the materials were the same as in Example 1, with Shigella dysenteriae type I, Shigella sonneri, Shigella flexneri 4 and Shigella baumannii 16 Antibodies from the monovalent diagnostic serum were used to coat reaction plates, and each antibody was coated with 4 wells, which were used to capture 4 kinds of Shigella, and the rest of the steps were the same as in Example 1. The PCR product identification results showed that there was no cross-reaction between species (see Table 2).

[0036]Table 2: Results of the crossover experiment for the detection of Shigella dysenteriae type I, Shigella sonneri, Shigella flexneri 4 and Shigella baumannii 16 using iUPPCR captured by specific monovalent antibodies

[0037] Shigella dysenteriae type I Shigella sonneri Shigella flexneri 4 Shigella baumannii 16 An...

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Abstract

The invention relates to a method of general-purpose primer PCR that is an immune catching method for detecting bacteria. Coating is carried out based on idiosyncratic antibody of various bacteria. With it being closed, the coated antibody is added into the bacteria solution at 36-38 deg.c for immune catching. Then heat denaturation releases template, and cracking liquid is extracted as PCR reaction template. Finally, using general-purpose primer of bacteria gene 16S rRNA to carry out PCR. The invention combines specificity of antigen-antibody with PCR, which has powerful rate of increasing production, to build iUPPCR for detecting pathogenic bacteria. The invention can catch idiosyncratic bacteria and increase caught bacteria so as to reach the goal for rapid detecting idiosyncratic bacteria. The invention possesses features of good repetitiveness, high sensibility and better specificity. It can detect almost any bacterium.

Description

(1) Technical field [0001] The invention relates to a universal primer PCR method for the immune capture method for detecting bacteria. (2) Background technology [0002] Bacterial infectious diseases have long endangered human health, and rapid and sensitive detection of pathogenic bacteria is an important step in disease prevention and control. In recent years, PCR technology has been applied to the detection of pathogenic bacteria. These detection technologies can be divided into two categories, namely PCR with specific primers and PCR with non-specific primers. The former often requires different primers depending on the target bacteria, while different primers The sequence may require different amplification conditions. Therefore, when the bacteria to be tested are unknown, multiple PCRs are required to obtain the results. Therefore, it is difficult to achieve the purpose of rapid and specific detection; the latter is generally synthesized based on the conservation of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/569
Inventor 彭宣宪王三英张建营
Owner XIAMEN UNIV
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