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Target gene for detecting citrobacter freundii and detection kit

A technology of Citrobacter freundii and a kit, which is applied in the field of molecular biology, can solve problems such as the lack of reports on the fluorescent quantitative PCR detection method of Citrobacter freundii, and achieve the purpose of avoiding non-specific amplification, reducing detection costs, The effect of high sensitivity

Inactive Publication Date: 2013-04-03
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the application of fluorescent TaqMan PCR technology, the choice of probe is an important factor, because it involves matching with primers and the most critical detection specificity
[0006] At present, in domestic and foreign research, there is no report on the fluorescent quantitative PCR detection method for Citrobacter freundii

Method used

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  • Target gene for detecting citrobacter freundii and detection kit
  • Target gene for detecting citrobacter freundii and detection kit
  • Target gene for detecting citrobacter freundii and detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 Determination of the specific DNA sequence of Citrobacter freundii

[0041] The present invention repeatedly compares and screens the TTRP gene of Citrobacter freundii and the nucleotide sequences of other bacteria published on NCBI, and finds that the TTRP gene of Citrobacter freundii only has higher homology with Salmonella, The homology reaches 84%, and its nucleotide sequence is shown in SEQ ID NO.1. Moreover, the gene has high specificity between 100bp and 650bp and is highly conserved, and can be used to distinguish Citrobacter freundii from its close Salmonella and other species of the genus Citrobacter.

Embodiment 2

[0042] The design of embodiment 2 Citrobacter freundii specific primers and probe

[0043] After repeated screening and verification, the present invention obtains a pair of fluorescent quantitative PCR primers for detecting Citrobacter freundii and a TaqMan probe used in conjunction with the primers, which are synthesized by Shanghai Jikang Biological Company. The probe is 5' labeled with a fluorophore (FAM) and 3' labeled with a quencher (BHQ1).

[0044] The primer sequence that the present invention obtains is:

[0045] Upstream primer: GCAGGACAGGAAGCGTCTG,

[0046] Downstream primer: CAGCCGACCATCTTTTCATAGC.

[0047] The probe sequence sequence is:

[0048] (FAM)-AGCCGCTGAACGACTTCCAGACCA-(BHQ1). The total length of the specific fragment amplified by the above primers is 126bp, and its nucleotide sequence is shown in SEQ ID NO.5. After sequencing, it was confirmed that the sequence belonged to the TTRP gene of Citrobacter flexneri.

Embodiment 3

[0049] The construction of embodiment 3 Citrobacter freundii genome DNA plasmid standard

[0050] 1. Extraction of bacterial genomic DNA

[0051] Bacterial genomic DNA was extracted using Promega's DNA Purification Kit, operate according to the instructions. The purity and concentration of bacterial genomic DNA were determined by ultraviolet spectrophotometer, and the genomic DNA of Citrobacter freundii ATCC43864 was diluted to 3×10 with EasyDilution buffer 5 ~5×10 2 Concentration gradient in pg / ul, the negative control was diluted to 3×10 with EasyDilution buffer 5 pg / ul, and genomic DNA were aliquoted in small quantities and stored at -20°C for later use.

[0052] 2. Establishment of common PCR reaction system

[0053] Common PCR reaction 20μl system is as follows:

[0054]

[0055] PCR reaction conditions:

[0056]

[0057] After the reaction, 5 μl of the PCR product was electrophoresed on a 1.5% agarose gel, and the analysis results were observed on a gel ima...

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Abstract

The invention provides a target gene for detecting citrobacter freundii. A nucleotide sequence is shown in SEQ ID NO.1. The invention also provides a real-time fluorescent quantitative PCR (polymerase chain reaction) primer and a probe. The nucleotide sequences of the primer and the probe are respectively shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. A kit for detecting the citrobacter freundii is also provided by the invention. The detection method and the kit thereof have the advantages of being accurate to detect, high in sensitivity, strong in specificity, simple and rapid, and have good clinical specimen detection ability.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to primers and probes for detecting a target gene of Citrobacter freundii and detecting the gene, and the present invention also relates to using the primers and probes to detect the target gene of Citrobacter freundii. Detection kits. Background technique [0002] Citrobacter freundii belongs to the genus Citrobacter, Enterobacteriaceae. Gram-negative bacilli, facultatively anaerobic, with flagella, pili, non-spores, and non-capsulated. Citrobacter freundii exists widely in nature and is a parasite in the intestinal tract of humans and animals. It has been reported in the literature that it can cause primary and secondary infections. C. freundii can be isolated from normal human and animal feces and the environment. However, researchers are increasingly aware that C. freundii can cause diarrhea and other infections such as urinary tract infections, meningitis, sepsis, and hospi...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/11C12Q1/68C12Q1/04C12R1/01
Inventor 刘丽云金东王艺婷徐建国叶长芸
Owner ICDC CHINA CDC