Specific primer and probe for real-time fluorescent PCR detection of enterococcus faecalis

A technology of Enterococcus faecalis and real-time fluorescence is applied in the field of microbial detection, which can solve the problems of fuzzy results and unsatisfactory results.

Inactive Publication Date: 2016-05-25
SUZHOU BAIYUAN GENT CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, various studies have shown that phenotype-based methods often yield ambiguous results because of phenotypic and biochemical similarities between multiple Enterococcus species and that phenotypic methods take days to produce results
The results of detecting EF by immunoenzyme test, immunodiffusion method, latex agglutination test, immunofluorescence method and enzyme-linked immunosorbent assay are not satisfactory

Method used

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  • Specific primer and probe for real-time fluorescent PCR detection of enterococcus faecalis
  • Specific primer and probe for real-time fluorescent PCR detection of enterococcus faecalis
  • Specific primer and probe for real-time fluorescent PCR detection of enterococcus faecalis

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Embodiment 1

[0045] The preparation of embodiment 1EF-16S gene standard product

[0046] To establish a real-time fluorescent quantitative PCR method, the external standard required by the method must first be prepared. The standard should contain highly conserved and specific sequences, and high specificity of the reaction must be ensured. The 16SRNA gene is highly conserved. In this study, the 16SRNA gene of Enterococcus faecalis was used as the target sequence. This example mainly uses PCR technology to amplify the 16S RNA gene of Enterococcus faecalis, and uses gene recombination technology to connect it to the plasmid vector pMD18-T to construct the recombinant plasmid pMD18-T-EF-16S, and carry out corresponding PCR identification and sequencing Identification, and finally quantified as a standard for the method to be established, laying the foundation for the next method and evaluation.

[0047] 1. Preparation of template DNA

[0048] Extract the genomic DNA of Enterococcus faecal...

Embodiment 2

[0121] Example 2 Establishment of real-time fluorescent quantitative PCR detection method for Enterococcus faecalis

[0122] 1. Design and synthesis of specific primers and probes

[0123] Taking the conserved fragment of the 16SRNA gene of Enterococcus faecalis selected above as the target, a set of real-time fluorescent quantitative PCR primers and probes were designed using Primerexpress3 software, PrimerPremier5 software and Oligo7 software.

[0124] As the core of the present invention, a group of primers and probe nucleotide sequences for the detection of Enterococcus faecalis real-time fluorescent PCR are as follows:

[0125] Upstream primer: EF-16S-140F5'-TTCGCACCAGGAGATAAA-3'

[0126] Downstream primer: EF-16S-252R5'-GCTGAATTGGCAGAGGAC-3'

[0127] Probe: EF-16S-168P5'-TTTTTCCCAGTTCACTGCCG-3'

[0128] The fluorescent reporter group at the 5' end of the probe is FAM, and the fluorescent quencher group TAMRA is labeled at the 3' end. At the same time, other fluorescen...

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Abstract

The invention provides a specific primer and probe for real-time fluorescent PCR detection of enterococcus faecalis. The specific primer and probe for real-time fluorescent PCR detection of the enterococcus faecalis are characterized in that the nucleotide sequences of the specific primer and probe is one of (1) or (2), for (1), an upstream primer is 5'-TTCGCACCAGGAGATAAA-3', a downstream primer is 5'-GCTGAATTGGCAGAGGAC-3', the probe is 5'-TTTTTCCCAGTTCACTGCCG-3', and the amplified target nucleotide sequence of the primer and probe are shown in SEQ ID No.3 in a sequence list; (2) is the sequence which is complementary to the sequence of (1). The specific primer and probe for real-time fluorescent PCR detection of the enterococcus faecalis can conduct qualitative and quantitative analysis on the clinically infected enterococcus faecalis content, has important significance on judging the clinically infected enterococcus faecalis content and plays an important role in the field of clinical tests.

Description

technical field [0001] The invention belongs to the technical field of microbial detection methods, and in particular relates to a set of specific primers and probes for real-time fluorescent PCR detection of Enterococcus faecalis. Background technique [0002] Enterococcus is a normal resident bacterium in human and animal intestines, and it is an important pathogenic bacterium of nosocomial infection. Among the infections caused by Gram-positive cocci, it ranks second. Enterococcus faecalis (Enterococcus faecalis) EF is a Gram-positive, catalase-negative coccus, which is the main flora in the intestinal tract of humans and animals. The bacterium can cause mass pathogenicity and hospital cross-infection, clinically manifested as urinary tract infection, bacteremia, endocarditis, etc. s concern. In human medicine, 85% to 90% of enterococcal infections are attributed to Enterococcus faecalis. Similarly, the impact of Enterococcus faecalis on animal breeding cannot be igno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/107C12Q2561/113
Inventor 车团结沈颂东尤崇革谢小冬李琳李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
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