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Method for detecting Fusobacterium nucleatum by using PCR-ELISA

A fusobacterium nucleatum, purpose technology, applied in the field of molecular biology detection, can solve the problems of low sensitivity, difficult to master, low cost, etc., and achieve good repeatability

Inactive Publication Date: 2020-05-26
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of F.nucleatum is often based on PCR, including conventional PCR and multiplex PCR technology. Conventional PCR technology is easy to operate and widely used, but conventional PCR needs to be stained with ethidium bromide, which has certain risks. Moreover, The electrophoresis results are simply observed by naked eyes, and the sensitivity is not strong; multiplex PCR technology requires the design of multiple pairs of primers, which is difficult to master
In addition, Loop-mediated isothermal amplification technology (Loop-mediated isothermal amplification, LAMP) is an emerging amplification technology, using LAMP to detect Fusobacterium nucleatum, the cost is low, but the requirements for primers are relatively high, and the scope of application is small. The problem of false positives is also more serious

Method used

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  • Method for detecting Fusobacterium nucleatum by using PCR-ELISA
  • Method for detecting Fusobacterium nucleatum by using PCR-ELISA
  • Method for detecting Fusobacterium nucleatum by using PCR-ELISA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The PCR amplification of embodiment 1FadA gene

[0029] According to the FadA gene of F. nucleatum in GeneBank, a pair of specific primers were designed and synthesized by Shanghai Bioengineering Co., Ltd.

[0030] The primer sequences are as follows:

[0031] F: 5'-CACAAGCTGACGCTGCTAGA-3'; SEQ ID NO.1;

[0032] R: 5'-TTACCAGCTCTTAAAGCTTG-3'; SEQ ID NO.2.

[0033] The freeze-dried powder of F. nucleatum was activated, operated in an anaerobic box with tryptone soybean liquid medium, the bacterial liquid was collected, the genomic DNA of F. nucleatum was extracted, and the FadA gene was amplified by PCR using it as a template.

[0034] Wherein, the PCR reaction system is: template 2 μl, upstream primer 0.5 μl, downstream primer 0.5 μl, Premix Taq 25 μl, double distilled water 22 μl.

[0035] The PCR reaction conditions were: 94°C for 5min; 30 cycles of 94°C for 30s, 59°C for 30s, and 72°C for 30s; 72°C for 10min; and storage at 4°C.

[0036] Use 1.5% agarose gel to c...

Embodiment 2

[0037] The construction of embodiment 2 recombinant plasmids

[0038] The amplified target gene was connected to the PMD-18-T vector, and transformed into DH5α cells to construct a recombinant plasmid; the constructed recombinant plasmid was sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the whole genome sequence of Fusobacterium nucleatum FadA was uploaded from NCBI. And compared with DNAman sequence analysis software, the result showed that the identity was 100% ( figure 2 ), consistent with the expected results, the target fragment size is 232bp.

Embodiment 3

[0039] Embodiment 3PCR-ELISA detects

[0040](1) The primers in Example 1 are labeled, wherein the 5' end of the upstream primer F is labeled with digoxin, and the 5' end of the downstream primer R is labeled with biotin; the recombinant plasmid constructed in Example 2 is used as a template, according to The conditions of Example 1 were used for PCR amplification, and then for ELISA detection.

[0041] (2) Dilute the streptavidin stock solution (1mg / ml) with carbonate buffer solution with pH 9.5, and add 100μl per well to the microtiter plate to coat, each sample is repeated three times, overnight at 4°C ; Wash 3 times with PBST, 3 min each time; Block with 2% BSA, 200 μl per well, place at 37°C for 2 h, wash the plate 3 times as well; Dilute the amplified product using the recombinant plasmid as template with PBST, 100 μl well, place at 37°C for 1 hour, wash the plate 4 times, 3 minutes each time; dilute horseradish peroxidase-labeled anti-digoxigenin antibody with PBST, 10...

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Abstract

The invention discloses a method for detecting Fusobacterium nucleatum by using PCR-ELISA for the purpose of non-diagnostic treatment, and belongs to the technical field of molecular biological detection. The method for detecting Fusobacterium nucleatum by using PCR-ELISA for the purpose of non-diagnostic treatment adopts a streptavidin-biotin-digoxin-anti-digoxin antibody system, so the detectionsensitivity is 100 times that of the conventional PCR, cross reactions with other bacteria such as Staphylococcus aureus, Clostridium botulinum and Salmonella are avoided, the specificity is high, the inter-batch and intra-batch variable coefficients are lower than 10%, and the Clostridium nucleatum PCR-ELISA detection method is high in sensitivity, specificity and repeatability.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a method for detecting Fusobacterium nucleatum by PCR-ELISA. Background technique [0002] Fusobacterium nucleatum (F.nucleatum) is a Gram-negative, non-spore-forming anaerobic bacterium. It is named Fusobacterium because of its enlarged middle and tapered ends like a shuttle. F.nucleatum is not easy to cultivate and has high requirements on the environment. It needs to create a strict anaerobic environment during cultivation. Generally, it grows better in an anaerobic box containing 5% hydrogen, 5% carbon dioxide, and 90% nitrogen. In addition, it has strict nutritional requirements. Generally, anaerobic blood plates are used for culture. After 48 hours, round, uneven surface, raised middle, translucent colonies with a diameter of about 1-2 mm are formed, emitting a foul smell. Leeuwenhoek, the father of microscopy, first discovered F. nucleatum in dental pl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04G01N33/543C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686G01N33/543
Inventor 刘兴友李鹏王瑞飞刘金晶王利平张艳芳魏小兵刘明成刘长忠谭东鹤
Owner XINXIANG UNIV
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