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Fusarium disease pathogenic bacteria RT-PCR (reverse transcription-polymerase chain reaction) detection primer and probe combination and kit

A technology for fusarium disease and detection primers, applied in the field of microbial detection, can solve the problems of not being suitable for early clinical diagnosis, unable to identify fungi to the level of species, time-consuming, etc., and achieve the effect of fast and convenient detection

Active Publication Date: 2017-06-20
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, except for culture, none of them can reach the level of fungal identification to the species level.
However, fungal culture takes too long, usually 1-2 weeks, and is not suitable for early clinical diagnosis

Method used

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  • Fusarium disease pathogenic bacteria RT-PCR (reverse transcription-polymerase chain reaction) detection primer and probe combination and kit
  • Fusarium disease pathogenic bacteria RT-PCR (reverse transcription-polymerase chain reaction) detection primer and probe combination and kit
  • Fusarium disease pathogenic bacteria RT-PCR (reverse transcription-polymerase chain reaction) detection primer and probe combination and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] The design of embodiment 1 primer, probe

[0078] So far, no real-time PCR detection method for the pathogen of corneal fusarium disease has been found. Therefore, combined with the NCBI database information and the inventor's previous sequence information on pathogenic Fusarium, compared with the seqman software, on large-subunit (LSU) rDNA and beta-tubulin, a general-purpose gene suitable for detecting Fusarium was found. Primer probes and their various specific primer probes ( figure 1 ). Major species include Fusarium solani, Fusarium moniliforme and Fusarium oxysporum. At the same time, the differences between Fusarium sequences and other common fungal sequences were fully considered.

[0079] Primer selection software was used for primer screening, supplemented by empirical manual correction, and regions without base variation were selected as amplification primers. After preliminary selection of primers, the Tm estimation formula using the concentration-depen...

Embodiment 2

[0116] Embodiment 2 is used for the kit of real-time fluorescence quantitative PCR detection fusarium pathogenic bacteria

[0117] The kit (20 servings) contains:

[0118] 1) DNA extraction tubes (20 pcs)

[0119] The extraction tubes are 2ml centrifuge tubes, each containing 600 μl of DNA extraction solution and 0.5 g of acid-washed glass beads (710-1180 μm in diameter, sigma, catalog number G1152). The formula of fungal DNA extraction solution is as follows:

[0120]

[0121] 2) PCR mixture A (2× master mix, 300μl), the formula is as follows:

[0122]

[0123] 3) PCR mixture B (260μl), the formula is as follows:

[0124]

[0125] 4) PCR mixture C (260μl), the formula is as follows:

[0126]

[0127] 5) PCR mixture D (260μl), the formula is as follows:

[0128]

[0129] 6) PCR mixture E (260μl), the formula is as follows:

[0130]

[0131] 7) Positive control (40 μl) 3 tubes, respectively 1 ng / μl of Exophiala dermatitidis, Exophiala zhenzi and Phialadium...

Embodiment 3

[0148] Embodiment 3 kit effect detection

[0149] 1) Standard curve creation and sensitivity detection

[0150] ① Construct plasmid standard

[0151] a. Amplify a single fragment with four pairs of primers, Fpan-F and Fpan-R, Fmon-F and Fmon-R, Fsol-F and Fsol-R, Foxy-F and Foxy-R respectively (completed with TAKARA's PremixTaqTM , Cat. No.: R004A), connected into the pUC18DNA plasmid vector (TAKARA, Cat. No.: 3218), and transformed into E.coli DH5α (TAKARA, Cat. No.: 9057). Positive clones were screened out by PCR detection, and purified plasmids were extracted and purified by alkaline lysis after shake culture.

[0152] b. Detect the plasmid concentration with Qubit 3.0 Fluorometer (Thermo Fisher Scientific), and calculate the copy number of the plasmid. The calculation formula is as follows:

[0153]

[0154] c. Diluted into different solutions containing the following copy numbers per microliter, the copy numbers are: 10 5 、10 4 、10 3 、10 2 , 10, 1.

[0155] ② C...

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Abstract

The invention provides fusarium disease pathogenic bacteria RT-PCR (reverse transcription-polymerase chain reaction) detection primer and probe combination and a kit. A nucleotide sequence of the primer and probe combination is shown as SEQ ID NO:1-12. The kit disclosed by the invention can quickly, specifically, flexibly and stably perform molecular diagnosis on cornea fusarium disease pathogenic bacteria. Based on the establishment of a reliable fusarium disease pathogenic bacteria detecting system, a competitiveness internal reference is pertinently designed to guarantee that negative results are genuine and believable. Meanwhile, the kit provided by the invention includes an improved DNA extracting system, can quickly and effectively finish a DNA extracting process and enables detection to be quicker and more convenient.

Description

technical field [0001] The invention belongs to the technical field of microorganism detection, and in particular relates to a real-time fluorescent quantitative PCR (RT-PCR) detection primer and probe combination and a kit for fusarium disease pathogens. Background technique [0002] Fungal keratitis is a common blinding eye disease all over the world, and it is more common in developing countries, including China, where agricultural populations dominate. Among them, corneal fusarium disease caused by Fusarium fungi is the most common. Statistics show that among the 775 cases of fungal keratitis reported by the Eye Institute of Tongren Hospital, Fusarium cornea accounted for 58.7%, and the pathogenic bacteria were mainly Fusarium solani, Fusarium moniliforme and Fusarium oxysporum. [0003] In recent years, with the widespread use of broad-spectrum antibiotics, corticosteroids, immunosuppressants and antineoplastic drugs, the incidence of corneal fusarium disease has a cle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/77
CPCC12Q1/6851C12Q1/6895C12Q2531/113C12Q2545/107
Inventor 龚杰张雯吴伟伟陈见友陆捷洁韩娜张婷婷黎青山
Owner ICDC CHINA CDC
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