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Utilizing tem-pcr technology to detect a variety of pathogenic bacteria by pcr detection universal primer pair

A technology of technical detection and universal primers, which is applied in the field of PCR detection of universal primer pairs, can solve problems such as false negatives, achieve good amplification results, balance amplification efficiency, and solve the effects of false negative results

Inactive Publication Date: 2015-08-19
哈尔滨海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only the concentration of super primers is sufficient for exponential amplification, which overcomes the amplification bias problem of multiplex PCR and solves the problem of false negative results caused by traditional multiplex PCR technology

Method used

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  • Utilizing tem-pcr technology to detect a variety of pathogenic bacteria by pcr detection universal primer pair
  • Utilizing tem-pcr technology to detect a variety of pathogenic bacteria by pcr detection universal primer pair
  • Utilizing tem-pcr technology to detect a variety of pathogenic bacteria by pcr detection universal primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of PCR template

[0045] According to the instruction manual of TIANamp Bacteria DNA Kit, various bacteria shown in Table 1 below were used as samples to extract and purify bacterial genomic DNA.

Embodiment 2

[0047] Primer design and synthesis

[0048] 1. Design and synthesis of non-homologous universal primers

[0049] Synthesize a pair of non-homologous universal primers (or super primers) to ensure that the pair of primers have no homology with bacteria and are non-homologous unique sequences. The primer sequences are as follows:

[0050] Super-F: AACTGTGTTTACTACTAGG;

[0051] Super-R: GGATTTAATGGCTACTTCTC.

[0052] 2. Design and synthesis of specific primers

[0053] Use the NCBI website and refer to relevant literature to select genes in the conserved regions of pathogenic bacteria.

[0054] Use the dnaman software to compare the nucleotide sequences of the conserved regions of three pathogenic bacteria, Enterohemorrhagic Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella, to find out the nucleotide sequences that are more suitable for designing specificity sequence area. Determine the target gene of enterohaemorrhagic Escherichia coli O157:H7 is rfbE, the ...

Embodiment 3

[0065] Optimization of PCR Conditions for Detection of Various Pathogenic Bacteria by Tem-PCR

[0066]Firstly, several different concentrations of general primers and specific primers are set, and the concentration of primers is optimized through orthogonal experiments. Then set several pre-cycle numbers and post-cycle numbers, and optimize the Tem-PCR amplification cycle numbers through orthogonal experiments. Finally determine the PCR reaction conditions of Tem-PCR method detection multiple pathogenic bacteria as follows:

[0067]

[0068]

[0069] The PCR reaction conditions were: pre-denaturation at 94°C for 10 min; 94°C for 30 s, 62°C for 1.5 min, 10 cycles; 94°C for 30 s, 46°C for 30 s, 72°C for 30 s, a total of 35 cycles; 72°C for 5 min.

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Abstract

The invention provides a PCR (polymerase chain reaction)-detection-based universal primer pair for detecting multiple pathogens by using a Tem-PCR technique. The primers are disclosed as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table. Specific primers for detecting three pathogens, which are respectively disclosed as SEQ ID NO.3-SEQ ID NO.8 in the sequence table, are firstly designed and synthesized; a pair of universal primers, which are disclosed as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table, are designed to associate so as to form chimeric primers, and the chimeric primers and universal primers are utilized to perform PCR amplification to detect pathogens. The method is simple and quick, has favorable specificity, and provides a new method for detecting pathogens.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a pair of universal primers for PCR detection which utilizes Tem-PCR technology to detect various pathogenic bacteria. Background technique [0002] At present, the detection methods for pathogenic bacteria mainly include national standard method, enzyme-linked fluorescent immunoassay, polymerase chain reaction (PCR), gold standard test paper method, DNA probe technology, and LAMP constant temperature amplification method. The disadvantage is that the detection throughput is not high. Multiplex PCR method is the basic method to improve detection throughput. Many high-throughput technologies, such as gene chip method and high performance liquid chromatography, are based on multiplex PCR method. However, the design of primers for multiplex PCR technology is difficult, and the problem of unbalanced amplification efficiency often occurs, resulting in false ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/42C12R1/19C12R1/01
CPCC12Q1/6848C12Q1/686C12Q2531/113C12Q2537/143
Inventor 刘忠梅徐义刚李淑云李苏龙曲敏莫春生
Owner 哈尔滨海关技术中心
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