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Highly specific gene fragment of Cronobacter spp. and its application

A high-specificity technology of Enterobacter sakazakii, applied in the field of molecular biology, can solve the problems of poor specificity, low specificity, and large sequence differences of 16SrDNA genes, and achieve low false positive rate, high specificity, and good specificity sexual effect

Active Publication Date: 2012-12-12
WUXI ZODOLABS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In past studies, there have been some PCR detection techniques with the 16S rDNA gene of Enterobacter sakazakii as the target gene (Lehner A et al., Gene based analysis of Enterobacter sakazakii strains from different source and development of a PCR assay for identification.[J]Molecular and Cellular Probes,2006.20(1):11-17), fluorescent quantitative PCR detection technology Malorny B et al., Detection of Enterobacter sakazakii strains by real-time PCR[J].Journal of Food Protection. 2005,68(8):1623-1627) and fluorescent probe detection technology (Almeida C et al., Development and application of a novel peptide nucleic acid oribe for the specific detection of Cronobacter genomospecies(Enterobacter sakazakii) in powered infant formula[J ].Appl.Environ.Microbiol,2009,75(9):2925-2930), but it is generally believed that there are large sequence differences among the strains of Enterobacter sakazakii, and the specificity of the 16SrDNA gene is relatively poor, and there are still inconsistencies in specificity. high or low sensitivity

Method used

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  • Highly specific gene fragment of Cronobacter spp. and its application
  • Highly specific gene fragment of Cronobacter spp. and its application
  • Highly specific gene fragment of Cronobacter spp. and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Acquisition of Specific Gene Fragments

[0053] 1. Use 16S rDNA universal primers to amplify the target sequence with Enterobacter sakazakii genomic DNA as a template;

[0054] 2. Obtain detailed sequence information through sequencing, and use NCBI's Blast tool to compare and analyze the obtained sequences to obtain highly conserved fragments. The matching rate of Enterobacter sakazakii is as high as 98%, but not that of Enterobacter sakazakii Less than 80% is the standard;

[0055] 3. Select the sequences at both ends, and design specific amplification primers based on the criterion that the last base at the end of the primer only matches the sequence of Enterobacter sakazakii and does not match the sequence of non-Enterobacter sakazakii, and use the designed primers to match the sequence of Enterobacter sakazakii Genomic DNA of Enterobacter sakazakii and non-Enterobacter sakazakii was amplified and tested;

[0056] 4、对选取的不同序列的结果进行比较和选择,最终获得了对不同阪崎肠杆菌菌株均具有...

Embodiment 2

[0057] Embodiment 2: Nucleic acid probe specific verification

[0058] 1. Design specific nucleic acid probes:

[0059] Using the specific sequence obtained by screening in Example 1 as a template, a specific capture probe for Enterobacter sakazakii was designed. The sequence of the probe is as follows:

[0060] PB: 5'-TCGTGCTGCGAGTTTGAGAGACTCTGACACACCGCG-3' (SEQ ID NO: 2).

[0061] 2. Probe specificity detection verification:

[0062] The specific recognition and capture of nucleic acid probes were verified by Southern Blotting experiments. The detailed steps are as follows: 32 strains of Enterobacter sakazakii and 23 strains of non-Sakazakii Enterobacter (including common intestinal pathogenic bacteria and probiotics Bacteria), and the genomic DNA of each strain was extracted, and after heat denaturation at 95°C for 5 minutes, it was quickly placed in an ice-water bath (to treat it as a single strand); respectively, the genomic DNA treated by heat denaturation was spotted ...

Embodiment 3

[0064] Embodiment 3: PCR primer specificity verification

[0065] 1. Primer design:

[0066] Using the specific sequence obtained by screening in Example 1 as the target sequence as a template, Enterobacter sakazakii-specific amplification primers were designed, and the primer sequences were as follows:

[0067] CsppF1: 5'-TCGTGCTGCGAGTTTGAGAG-3' (SEQ ID NO: 3)

[0068] CsppR1: 5'-CCTCGCGTGCTCACACAG-3' (SEQ ID NO:4)

[0069] CsppF2: 5'-AGAGACTCTGACACACCGCG-3' (SEQ ID NO: 5)

[0070] CsppR2: 5'-AATGAGTGAAAGGCGTTACCG-3' (SEQ ID NO: 6)

[0071] 2. Verification of primer specificity

[0072] The specificity of primers CsppF1 / CsppR1 and CsppF2 / CsppR2 was verified by nested PCR amplification using the genomic DNA of the above 32 strains of Enterobacter sakazakii and 23 strains of non-Enterobacter sakazakii as templates. Proceed as follows:

[0073]The genomic DNA templates of each strain were used for the first round of nested PCR amplification. PCR reaction system (20 μL): 2 ...

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Abstract

The invention relates to a Cronobacter spp. 16S rDNA gene based sequence, and also provides a method for specific detection of Cronobacter spp. by combining magnetic beads and a nested PCR (polymerase chain reaction) technology. The advantage of the invention lies in that: a specific probe and primers of the Cronobacter spp. 16S rDNA gene sequence are utilized, and magnetic beads are combined to conduct rapid and specific capture and enrichment of a Cronobacter spp. target gene in a sample, finally amplification detection is performed through the nested PCR technology, and the detection sensitivity is improved.

Description

technical field [0001] The present invention relates to the field of molecular biology. More specifically, it relates to a detection method based on the 16S rDNA specific gene sequence of Enterobacter sakazakii (Cronobacter spp.), and also relates to a detection method based on this sequence and applying magnetic beads and nested PCR technology. Background technique [0002] Enterobacter sakazakii is a food-borne opportunistic pathogen that can cause severe neonatal meningitis, necrotizing colitis, and bacteremia, and can even lead to a high mortality rate and severe neurological sequelae , and developmental disorders (Nazarowecwhite M., J.M.Farber. Enterobacter sakazakii: A review [J]. International Journal of Food Microbiology, 1997, 34(2): 103-113.). According to clinical cases, the main infection groups of Enterobacter sakazakii are newborns and immunocompromised infants, as well as immunocompromised adults and the elderly, and the infection fatality rate is as high as ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12Q1/10
CPCY02A50/30
Inventor 李林杨晓慧许恒毅徐锋
Owner WUXI ZODOLABS BIOTECH
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