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Highly specific gene segment of Cronobacter spp. and application thereof

A Cronobacter, high specificity technology, applied in the field of molecular biology, can solve the problems of poor specificity, low specificity and large sequence difference of 16S rDNA gene, and achieves low false positive rate, high specificity and high specificity. sexual effect

Inactive Publication Date: 2013-12-25
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 for the target gene of the 16S rDNA gene of Cronobacter (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 the sequences of Cronobacter strains are quite different, and the specificity of the 16S rDNA gene is relatively poor, and there is still specificity Problems such as low or low sensitivity

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  • Highly specific gene segment of Cronobacter spp. and application thereof
  • Highly specific gene segment of Cronobacter spp. and application thereof
  • Highly specific gene segment of Cronobacter spp. and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Acquisition of Specific Gene Fragments

[0042] 1. Using 16S rDNA universal primers:

[0043] F341: 5'-CCTACGGGAGGCAGCAG-3' (SEQ ID NO. 6),

[0044] R518: 5'-ATTACCGCGGCTGCTGG-3' (SEQ ID NO. 7);

[0045] Using Cronobacter genomic DNA as a template, the target sequence was amplified. PCR reaction system (50 μl): 25 μl of 2×PFU mix, 1.5 μl of each primer V3F / V3R (1 μM), 2 μl of genomic DNA, and 20 μl of sterile water. The PCR reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 54°C annealing for 30 s, 72°C extension for 3 min, 20 cycles; 72°C final extension for 10 min.

[0046] 2. Obtain detailed sequence information through sequencing, and use NCBI's Blast tool to compare and screen the obtained sequences to obtain highly conserved fragments. The matching rate of Cronobacter is as high as 98%, but that of non-Cronobacter Less than 80% is the standard;

[0047] 3. Select the sequences at both ends, and use the last base ...

Embodiment 2

[0050] Example 2: PCR primer specificity verification

[0051] 1. Primer design:

[0052] Taking the specific sequence obtained by screening in Example 1 as the target sequence as a template, the Cronobacter specific amplification primers were designed, and the primer sequences were as follows:

[0053] VicF1: 5'-TCGTGCTGCGAGTTTGAGAG-3' (SEQ ID NO.2)

[0054] VicR1: 5'-CCTCGCGTGCTCACACAG-3' (SEQ ID NO.3)

[0055] VicF2: 5'-AGAGACTCTGACACACCGCG-3' (SEQ ID NO.4)

[0056] VicR2: 5'-AATGAGTGAAAGGCGTTACCG-3' (SEQ ID NO.5)

[0057] 2. Verification of primer specificity

[0058] Using the genomic DNA of 31 strains of Cronobacter and 21 strains of non-Cronobacteria as templates, the specificity of primers VicF1 / VicR1 and VicF2 / VicR2 was verified by nested PCR amplification detection. The results are shown in Table 1. The steps are as follows:

[0059] Take the genomic DNA template of each strain for the first round of nested PCR amplification, PCR reaction system (25 μl): 2×Taq m...

Embodiment 3

[0069] Example 3: Sample detection application of silica magnetic microspheres

[0070] 1. Detection of pure culture samples of different concentrations of Cronobacter

[0071] Using silica magnetic microspheres (Magpearl Silica, 0.3-0.5 μm, 10 mg / ml, purchased from Zhengzhou Inno Biotechnology Co., Ltd.) and nested PCR technology for specific detection of Cronobacter pure culture samples. The pure culture of Cronobacter was diluted with sterile 0.01M PBS gradient to obtain a concentration gradient of bacteria: 10 7 ,10 6 ,10 5 ,10 4 ,10 3 ,10 2 ,10 1 ,10 0 For the sample of CFU / ml, add 1ml of bacteria dilution solution of different concentrations to each 2ml centrifuge tube in turn as the sample to be tested, and add 1ml of sterile water to the 2ml centrifuge tube as a negative control. Place the centrifuge tube in a boiling water bath for 10 minutes to lyse the cells to release genomic DNA; add 700uL high salt solution (3M NaCl, 2M KCl) and 10uL silica magnetic micro...

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Abstract

The invention relates to a method for specific detection of Cronobacter spp. with silica magnetic microballoon and nested PCR technology based on a Cronobacter spp. 16SrDNA gene specific sequence, which is shown in the SEQID NO.1. Under specific condition, the property that silica magnetic microballoons can absorb nucleic acids is used for realizing rapid adsorption, enrichment and separation of pathogen nucleic acids in milk powder samples; two pairs of highly specific primers are designed based on Cronobacter spp. 16SrDNA gene, Cronobacter spp. specific gene is amplified by nested PCR technology, thereby realizing accurate, rapid and high sensitivity detection of Cronobacter spp. existed in the samples.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a specific gene sequence based on Cronobacter spp. 16S rDNA and its application. Background technique [0002] Cronobacter sakazakii (formerly known as Enterobacter sakazakii) is a food-borne opportunistic pathogen that can cause severe neonatal meningitis, enterocolitis, and bacteremia, which can lead to severe neurological sequelae and Developmental disorders (Nazaroecwhite 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 Cronobacter are neonates, premature infants and the elderly with low immunity, and the fatality rate of infection is as high as 20% to 50%. Currently, the source of Cronobacter contamination is still under study. According to epidemiological studies, most clinical cases indicated that the source of infection was infant formula. ...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12Q1/68C12Q1/04
Inventor 李林杨晓慧明星徐波
Owner WUXI ZODOLABS BIOTECH
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