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RNA isothermal amplification nucleic acid detection kit aiming at Escherichia coli 0157

A technology for O157 and Escherichia coli, which is applied to the determination/testing of microorganisms, fluorescence/phosphorescence, biochemical equipment and methods, etc., and can solve the problems of contamination experiment results, false positive and false negative detection costs, etc.

Active Publication Date: 2013-12-04
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of low sensitivity of the existing detection method for Escherichia coli O157 (O157), long detection cycle, easy to cause contamination of amplified products, false positive or false negative of experimental results and high detection cost, the present invention provides a A real-time fluorescent nucleic acid constant temperature amplification detection technology for Escherichia coli O157 (O157) that has short detection cycle, high sensitivity, high specificity, can avoid false positives, low pollution, stable reaction and low detection cost, and is easy to popularize and apply, including special primers , probes, kits and their use

Method used

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  • RNA isothermal amplification nucleic acid detection kit aiming at Escherichia coli 0157
  • RNA isothermal amplification nucleic acid detection kit aiming at Escherichia coli 0157
  • RNA isothermal amplification nucleic acid detection kit aiming at Escherichia coli 0157

Examples

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Effect test

Embodiment 1

[0103] Example 1. Design of special primers and probes for detection of Escherichia coli O157 (O157) by real-time fluorescent nucleic acid constant temperature amplification

[0104] In the present invention, no secondary structure and highly conserved segment in rfbE of O157 bacteria is selected as the amplified target sequence region (its nucleotide sequence is shown in sequence 1 in the sequence table), and according to the principle of primer probe design, DNA STAR, DNAman Software and artificially designed special primers and probe sequences for real-time fluorescent nucleic acid constant temperature amplification detection of Escherichia coli O157 (O157), and the following specific sequences were obtained:

[0105] (1) A capture probe (TCO, Target Capture Oligo) that can specifically bind to the target nucleic acid (O157RNA) sequence of Escherichia coli O157 (O157) shown in Sequence 1 in the sequence listing (TCO, Target Capture Oligo), the nucleoside of the capture probe...

Embodiment 2

[0109] Example 2. Preparation of a real-time fluorescent nucleic acid constant temperature amplification detection kit for Escherichia coli O157 (O157)

[0110] Using the special primers and probes provided in Example 1, a real-time fluorescent nucleic acid constant temperature amplification detection kit for Escherichia coli O157 (O157) of the present invention was obtained. The kit contains a capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, O157 detection probe, M-MLV reverse transcriptase and T7 RNA polymerase; when there is an internal standard in the kit, it also includes an internal standard Detection probe.

[0111] The capture probe exists in the nucleic acid extraction solution, the T7 primer, nT7 primer and O157 detection probe, the internal standard detection probe exist in the O157 detection solution, the M-MLV reverse transcriptase and T7 RNA polymerase It exists in the SAT enzyme solution. Specifically, the kit is divided into box A (specimen proc...

Embodiment 3

[0144] Embodiment 3, real-time fluorescent nucleic acid constant temperature amplification detection sensitivity of Escherichia coli O157

[0145] Use the kit of the present invention (see Example 2 for the composition, there is no VP internal standard in the kit, and there is no internal standard detection probe in the detection solution) to detect Escherichia coli O157 (O157) in food samples, and the concentration is 1× 10 7 CFU / mL positive reference substance, diluted to 10 by 10 times 2 CFU / mL, set up a negative control (0157 negative control, which does not contain the target nucleic acid (0157RNA) of E. coli O157). The specific method includes the following steps:

[0146] (1) Dilution of bacteria solution

[0147] The measured concentration is 1×10 7 CFU / mL Escherichia coli O157 culture, 10-fold serial dilution to 100CFU / mL as Escherichia coli O157 linear sensitivity reference.

[0148] (2) Nucleic acid extraction

[0149] 2.1 Add 200 μl lysate (containing HEPES 3...

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Abstract

The invention discloses an RNA isothermal amplification nucleic acid detection kit aiming at Escherichia coli 0157. The kit comprises a capture probe, a pair of 0157 detection primer T7 and n T7 primer, a 0157 detection probe, M-MLV reverse transcriptase enzymes, T7 RNA polymerases and other reagents. The kit provided by the invention can detect 0157 RNA in foods, has the characteristics of high specificity, high sensitivity (which can reach 103 CFU / mL), low pollution (as the amplification product RNA is susceptible to degradation in the natural environment), accurate result (as false positives can be avoided) and quick detection (as the detection can be completed within 40 minutes conventionally ), plays an important role in the quick detection of the Escherichia coli 0157, and has a wide application prospect.

Description

technical field [0001] The present invention relates to the biological detection technology of pathogenic bacteria, in particular to the primers and probes used in the real-time fluorescent nucleic acid constant temperature amplification detection of E. Needles and related kits. Background technique [0002] Escherichia coli O157, belonging to the family Enterobacteriaceae and the genus Escherichia, is one of the main intestinal pathogens. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 has been designated as a new foodborne pathogen by WHO. Can cause hemorrhagic enteritis, hemolytic uremic syndrome and thrombocytopenic purpura. Escherichia coli O157 infection is often closely related to raw beef, fresh milk or dairy products processed with fresh milk. Other known routes of transmission are raw milk, sausages, barbecued meats, unchlorinated municipal water, apple juice, raw vegetables, salads and mayonnaise. In addition, food may be contaminated by food handlers infect...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 尹华立于明辉张长明朱凤居金良
Owner SHANGHAI RENDU BIOTECH
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