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Primers for assaying citrobacter rodentium and kit thereof

A technology of Citrobacter bacillus and a kit, which is applied in the field of microbial detection, can solve the problems of unsatisfactory monitoring effect, limited popularization and application, easy pollution and the like, and achieves the effects of suitable popularization and application, high sensitivity, and low requirements for instruments and equipment.

Inactive Publication Date: 2018-05-29
SHANGHAI RES CENT FOR MODEL ORGANISMS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the monitoring effect of the culture method is not ideal for the bacteria.
Conventional molecular biology methods such as PCR have been widely used due to their advantages in rapidity, specificity, and sensitivity. However, the detection process requires the support of expensive amplification equipment and other equipment, and is prone to contamination and false positives. To some extent, this method is limited in the promotion and application of the first-line field

Method used

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  • Primers for assaying citrobacter rodentium and kit thereof
  • Primers for assaying citrobacter rodentium and kit thereof
  • Primers for assaying citrobacter rodentium and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Design of Primers for LAMP Detection of Citrobacter rodentium

[0045]Specific LAMP primers were designed for the conserved region of the esp B gene sequence of Citrobacter rodentium registered in GenBank, and the sequence is as follows:

[0046] F3 Forward Outer Primer: 5'-CCGCCTCTGAAGTTTCAACT-3'(SEQ ID NO.1)

[0047] B3 reverse outer primer: 5'-AAATCCTGAGCAGCGTCAG-3' (SEQ ID NO.2)

[0048] FIP forward inner primer:

[0049] 5'-CGCGATCCCTTGCTGCAATGTTTCCGAAAGTATTGCGGATGC-3' (SEQ ID NO.3)

[0050] BIP reverse inner primer:

[0051] 5'-AAGCGGCAAATCGTACATCAGGTGCCTTTTCAGCTACCTGAGAA-3' (SEQ ID NO.4)

[0052] LF forward loop primer: 5'-GCCAACTGTCTCATCTGCG-3' (SEQ ID NO.5)

[0053] LB reverse loop primer: 5'-TGTTACTACTTCTGCTCAGAAAGC-3' (SEQ ID NO.6)

Embodiment 2

[0055] Genomic DNA extraction and LAMP amplification system establishment

[0056] The genomic DNA of Citrobacter rodentium was extracted using the TIANGEN Bacterial Genomic DNA Extraction Kit, and the OD260 / 280 was measured by an ultraviolet spectrophotometer in the range of 1.6-2.0, and stored at -20°C for later use.

[0057] The LAMP reaction system is as follows:

[0058]

[0059] Optimization of the amplification temperature, the optimization results are as follows figure 1 shown.

[0060]

[0061] Select 60°C, 62°C, 64°C and 66°C as the amplification temperature to optimize the amplification temperature of the primers. The results showed that the water was not amplified, and the positive nucleic acid amplification efficiency was not much different. The peak time of positive nucleic acid at 60°C and 66°C was around 30 minutes, and the peak time of positive nucleic acid at 62°C and 64°C was slightly earlier, at around 27min. . Therefore, the intermediate temperat...

Embodiment 3

[0064] LAMP-specific analysis

[0065] Using Citrobacter rodentium genomic DNA as a positive control template, sterilized double distilled water as a negative control template, Salmonella typhimurium, Bordetella bronchus, Pasteurella pneumophila, Shigella flexneri, Staphylococcus aureus, Genomic DNA of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Corynebacterium bovis were used as experimental objects, and the LAMP amplification method established by the present invention was used to detect them respectively, so as to evaluate the specificity of the method.

[0066] The result is as Figure 2~4 as shown, figure 2 It is the LAMP amplification map of the specificity test; image 3 It is the visual detection result diagram of the specificity test, in which, 1 is Citrobacter rodentium, 2 is water, 3 is Salmonella typhimurium, 4 is Bordetella bronchus, 5 is Pasteurella pneumophila, and 6 is Shigella flexneri bacteria, 7 for Staphylococcus aureus, 8 for Pseudomonas aerugin...

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Abstract

The invention relates to a group of primers for assaying citrobacter rodentium and a kit thereof, which belong to the technical field of microbiological assay. The primers for assaying the citrobacterrodentium, provided by the invention, include an outer primer pair, an inner primer pair, and a loop primer pair; sequences of the outer primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2; sequences of the inner primer pair are shown as SEQ ID NO.3 and SEQ ID NO.4; and sequences of the loop primer pair are shown as SEQ ID NO.5 and SEQ ID NO.6. The specific primers provided by the invention havegood specificity and high sensitivity, and can rapidly assay the citrobacter rodentium.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a set of primers for detecting Citrobacter rodentium and a kit thereof. Background technique [0002] Citrobacter rodentium (C.rodentium) is one of the members of the genus Citrobacter in the Enterobacteriaceae family. Pathogenic Escherichia coli is a natural murine pathogen with the same Lee virulence island. The bacterium is an opportunistic pathogenic bacterium, which is mainly transmitted through fecal-oral transmission. The course of the disease is short, generally lasting about 4 weeks, and can cause infectious colonic hyperplasia in mice. Animals show rough coat, weight loss, growth retardation, diarrhea, Symptoms such as colitis and even rectal prolapse. Adult mice generally have no clinical symptoms, and unweaned or newly weaned pups are more susceptible than adult mice. [0003] The classic detection method for C. rodentium is isolation and culture, w...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/10C12N15/11C12R1/01
CPCC12Q1/689
Inventor 冯洁谢建芸高诚
Owner SHANGHAI RES CENT FOR MODEL ORGANISMS
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