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Primer for detecting pathogenic microorganism and multiple PCR using the same

A pathogenic microorganism and multiple amplification technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, recombinant DNA technology, etc., can solve the cumbersome and time-consuming problems, the inability to quickly and accurately diagnose, and the lack of specific high-quality diagnostic serum and other problems, to achieve broad application prospects and high specificity

Inactive Publication Date: 2010-10-06
ICDC CHINA CDC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the grassroots disease control units in my country still use the traditional detection method based on isolation, culture and biochemical identification, which is cumbersome and time-consuming, and can no longer meet the complex and changeable epidemic treatment work.
The use of immunological methods and rapid bacterial identification equipment has alleviated this situation to a certain extent, but due to the lack of specific and high-quality diagnostic serum, and the limitation of the number and throughput of diagnostic strains, it is still impossible to quickly and accurately identify bacteria. diagnosis

Method used

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  • Primer for detecting pathogenic microorganism and multiple PCR using the same
  • Primer for detecting pathogenic microorganism and multiple PCR using the same
  • Primer for detecting pathogenic microorganism and multiple PCR using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, intra-species and inter-species mixed primer PCR

[0052] 1. Template Preparation

[0053] Pick a single colony from a petri dish and inoculate it in a liquid medium for culture, take an appropriate amount of bacterial solution and put it in a boiling water bath for 10 minutes, centrifuge at 13000rpm for 10 minutes, and take the supernatant as a template.

[0054] 2. Selection of virulence genes and specific primers of enteric pathogens

[0055] The following genes were selected by consulting the literature and the GenBank database (see Table 2): rfbE, eaeA of EHEC, lt, st of ETEC, eaeA, bfpA of EPEC, ipaH of EIEC, o1ag, ct of Vibrio cholerae O1 serogroup Eltor biotype , rtx, o1ag, ct of O1classic, o139ag, rtx of O139 group, tl, tdh, trh of Vibrio parahaemolyticus, mapA, bp, cdt of Campylobacter jejuni, iroB, rfbS of Salmonella.

[0056] Primers were designed according to the conserved region of the gene, the length of the product was 300-500 bp, and the...

Embodiment 2

[0077] Embodiment 2, multiplex PCR incorporation of fluorescein control without the output of fluorescein

[0078] The difference in the yield of the amplified product was compared with and without the addition of fluorescein in the reaction system.

[0079]Reaction system: 25μl, 2.5μl of 10×PCR buffer, 0.5μl of three dNTPs (10mM, without dTTP), 4μl of dTTP (1mM), 0.5μl of cy5-dUTP (1mM), 1u of taq enzyme (2u / μl), Primer (1.6 μM) 2.5 μl, template 0.5 μl, deionized water 14 μl. The reaction conditions are the same as single weight.

[0080] Results: The incorporation of fluorescein in the reaction system significantly reduced the amplification yield (see Figure 2A with Figure 2B ).

Embodiment 3

[0081] Embodiment 3, the making and hybridization of gene chip

[0082] Use spotting instrument (GeneTAC Microarraying, Perkin Elmer Company, USA) to spot specific virulence gene fragments in a certain order on amino-modified glass slides, including six 6×6 matrices, each matrix consists of four positioning coordinate points and four probes, each probe longitudinally replicated four points. The chips were fixed by rehydration, UV cross-linking and high-temperature dry-baking. Dot matrix composition see Figure 3A . Put the processed chip into a GenTAC Hybridization hybridization instrument, add 100 μl of pre-hybridization solution, agitate at 42° C. for 40 minutes, and wash with 0.1% SDS. Take 20 μl of the purified labeled sample, add 80 μl of hybridization solution, mix well, inject into the hybridization instrument, cover the chip probe sample array, hybridize at 42°C for 12 hours, wash unbound probes with cleaning solution, desalt, and dry in the air. The scanner reads ...

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Abstract

The invention relates to a primer used for detection of pathogenic microorganism so as to detect pathogenic microorganism such as vibrio cholerae, pathogenic escherichia coli, campylobacter jejuni, Yersinia enterocolitica, parahemolytic vibrio, salmonella, shigella and Listeria monocytogenes, etc. The invention also relates to a method by using the primer to do multiple amplification detection and further relates to applications of the primer for detection of pathogenic microorganism in preparation of detection agent and further relates to kits containing the primer for the detection of pathogenic microorganism.

Description

[0001] The invention is based on the following funded subject [0002] 1. Topic 1: National Natural Science Foundation of China Approval Number: 30170052 "Molecular Biology of Campylobacter jejuni Associated with Guillain-Barré Syndrome" 2002.01-2004.12; [0003] 2. Topic 2: Social Welfare Project of the Ministry of Science and Technology: "Establishment of National Rapid Inspection Method System for Imported and Exported Food" 2003; Project No.: 2002DIA50036; [0004] 3. Topic 3: Social welfare project of the Ministry of Science and Technology: "Establishment of a rapid detection system for pathogens and genetically modified labels in food" 2005; project number: 2004DIB2J065. technical field [0005] The invention relates to the field of biotechnology, in particular to primers for detecting pathogenic microorganisms and multiple amplification using the primers. Background technique [0006] Infectious diseases caused by microorganisms are still the main diseases threateni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12P19/34
CPCY02A50/30
Inventor 张建中曾浔尤元海姜海肖迪闫笑梅
Owner ICDC CHINA CDC
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