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Cronobacter sakazakii O antigen specific nucleotides and use thereof

A technology of Enterobacter sakazakii and nucleotides, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., and can solve the problems of difficult preparation and storage of antiserum, incomplete types of antiserum, and poor accuracy , to achieve the effects of short detection cycle, easy industrial production, and high speed

Active Publication Date: 2011-08-17
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This diagnostic method requires a large amount of antiserum, and the antiserum is generally incomplete and insufficient in quantity, and there are some difficulties in the preparation and storage of a large amount of antiserum
On the other hand, this method takes a long time, has low sensitivity, high missed detection rate, and poor accuracy. There are often cross-reactions between antisera produced by different O antigens.

Method used

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  • Cronobacter sakazakii O antigen specific nucleotides and use thereof
  • Cronobacter sakazakii O antigen specific nucleotides and use thereof
  • Cronobacter sakazakii O antigen specific nucleotides and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 : Genome Extraction

[0035] Enterobacter sakazakii was cultured in LB liquid medium at 37°C, the bacteria were collected, and the genome was extracted. The specific steps are as follows:

[0036] The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg / ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul 20mg / ml proteinase K, 15ul 10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg / ml RNase, and incubate at 65°C for 30 minutes. Add an equal volume of phenol to extract the mixture, take the supernatant, and then extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether. Extract to remove residual phenol. The supernatant was precipitated with 2 times the volume of ethanol, the DNA was rolled out with glass wool and washed with 70% ethanol, and finally the DNA w...

Embodiment 2

[0037] Example 2: sequence deciphering

[0038] (1) The O antigen gene cluster of Enterobacter sakazakii was amplified by Long-PCR. According to the JumpStart between the galF gene and the O antigen gene cluster in Genbank, the upstream primer was designed as wl-10324, 5'-GCACTGGTAGCTATTGAGCCAGGGGCGGTAGCAT-3', and the downstream primer was designed as wl-22115'-ACTGCCATACCGACGACGCCGATCTGTTGCTTGG-3' according to the gnd gene (respectively SEQ ID NOS: 15-16).

[0039] The PCR reaction program is as follows: pre-denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 61°C for 30 seconds, and extension at 68°C for 15 minutes, so that 30 cycles were performed, and finally, extended at 68°C for 8 minutes to obtain PCR For the product, the size and specificity of the PCR product were detected by 0.8% agarose gel electrophoresis, 8 tubes of 50 μl long PCR product were combined, and the PCR product was purified with the Wizard PCR Preps purification ...

Embodiment 3

[0043] Example 3 : Primer design and screening

[0044] According to the deciphering of the gene cluster, we found that the wzy and wzx genes are indeed serotype-specific genes, so we selected the specific segments of the genes to design specific primers. Since wzy is more specific, the wzy gene is mainly used as the target gene, but because the gene is not contained in the O6 serotype, primers are designed for this serotype with wzx as the target gene.

[0045] Primer design is a core part of this invention. Primers were designed according to the specific genes described in the literature. The two genes wzx and wzy are relatively specific genes in the O antigen gene cluster of Enterobacter sakazakii, which can be used as target genes for serotype identification. The above-mentioned genes were introduced into Primer Premier 5 for primer design. The length of the primers should preferably be between 18-24 bp, and the Tm value should be between 50-55°C. A pair of primers ar...

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Abstract

The invention relates to a Cronobacter sakazakii O antigen specific nucleotides and use thereof. The nucleotides comprises: 1) at least one of nucleotides represented by sequences from SEQ ID No.1 to SEQ ID No.14; and 2) at least of nucleotides complementary to the nucleotides represented by the sequences from SEQ ID No.1 to SEQ ID No.14. The nucleotides can be used for preparing polymerase chain reaction (PCR) kits and gene chips for detecting Cronobacter sakazakii. The Cronobacter sakazakii O antigen specific nucleotides, and the PCR kits and the gene chips containing the nucleotides have high practicality; the preparation method of the PCR kits are simple and convenient, the detection period is short, the detection speed is high, the operability of the kits is high, the industrial production of the kits is easy, and the detection cost is relatively low; the accuracy is high; and the sensitivity is high.

Description

technical field [0001] The invention relates to a nucleotide specific to the O antigen gene cluster of Enterobacter sakazakii and application thereof, in particular to a nucleotide specific to a single gene in the O antigen gene cluster of Enterobacter sakazakii and the application thereof. Background technique [0002] Enterobacter sakazakii is an intestinal parasite and opportunistic pathogen of humans and animals. It has been identified by the World Health Organization and many countries as an important conditional pathogen that causes infant mortality. It can cause diseases in any age group, especially It is the greatest threat to premature infants, low birth weight infants, or immunocompromised infants. In severe cases, it can lead to sepsis, meningitis, or necrotizing enterocolitis, and the mortality rate can reach 40% to 80%. At present, microbiologists do not know the source of the contamination of Enterobacter sakazakii, but many research reports show that infant fo...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/10
Inventor 王磊何欣王敏孙亚民曹勃阳
Owner TIANJIN BIOCHIP TECH CO LTD
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