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Fluorescent detection kit of cholera vibrio O139 and detection method

A technology for Vibrio cholerae and fluorescence detection, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc. It can solve the problems of inaccurate detection results, false positives, long cycle and so on, so as to reduce false positives Rate, Sensitivity Reduction, Sensitivity Improvement Effects

Inactive Publication Date: 2009-04-29
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] The traditional methods for detection of Vibrio cholerae mainly include morphology, biochemistry, coagulation test, etc. The cycle of these detections is long, generally more than 8 to 10 hours. strains) cannot be detected
Although some immunological methods are fast, the results are unstable, and they can only be used for rapid preliminary screening of suspected patients, and cannot be used as a basis for diagnosis
Although PCR technology can quickly diagnose pathogenic bacteria, there are a large number of false positive results.
Studies have shown that Vibrio cholerae O1 and O139 are highly similar in many gene structures and compositions; some virulence factor genes such as ctxA and tcpA are shared by the two serotypes, so only the above-mentioned virulence genes The test result is inaccurate; however, there is a large difference in the sequence of the glycosyltransferase gene (LPSgt) of the two serotypes of Vibrio cholerae through comparison

Method used

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  • Fluorescent detection kit of cholera vibrio O139 and detection method
  • Fluorescent detection kit of cholera vibrio O139 and detection method
  • Fluorescent detection kit of cholera vibrio O139 and detection method

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Embodiment 1

[0030] Embodiment 1: the acquisition of specific primers, fluorescent probes and standards

[0031] 1. Materials:

[0032]Bacterial genomic DNA extraction reagents were purchased from Dalian Bao Biological Engineering Co., Ltd.; restriction endonucleases were purchased from LTI / Gibco, USA; pGEM-T-Easy cloning system, PCR buffer, and TaqDNA polymerase were purchased from Promega, USA; sequencing reagents, Model 377 sequencer, Bio-Radicycler PCR instrument and Model 7000 quantitative PCR instrument are all products of American ABI Company.

[0033] 2. Primer and probe design and synthesis:

[0034] Using the Vibrio cholerae O139 glycosyltransferase gene sequence (registration number U72485) as a template, use Primer Express TM (V2.0, American ABI Company) software analyzes TaqMan primer and probe site, selects the optimal combination therefrom.

[0035] Standard PCR upstream primer sequence is:

[0036] 5'-TGTAATGATT AGAAAGAAAG TTA-3',

[0037] Downstream primers are:

[0...

Embodiment 2

[0061] Embodiment 2: Fluorescent quantitative PCR method detects Vibrio cholerae O139

[0062] 1. Specimen detection:

[0063] 52 cases of clinical stool samples were washed with normal saline and then centrifuged. Genomic DNA was extracted with genomic DNA extraction reagents. 1.0 μL was taken as a template, and PCR amplification was carried out on a 7000 quantitative PCR instrument of ABI Company with upstream and downstream primers for detection.

[0064] The composition of the PCR reaction solution is as follows:

[0065] 2×PCR buffer 10.0μL

[0066] Detection upstream primer (10μM) 1μL

[0067] Downstream primer for detection (10μM) 1μL

[0068] Fluorescent probe for detection (10μM) 1μL

[0069] Taq DNA polymerase (5U / μL) 0.2μL

[0070] dNTPs (250mM each) 1.60μL

[0071] (dATP, dTTP, dCTP, dGTP substance ratio 1:1:1:1)

[0072] Template DNA (50ng / μL) 1μL

[0073] Make up to 20 μL with water.

[0074] The PCR reaction conditions were: pre-denaturation at 93°C for...

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Abstract

The invention provides a fluoroscopic examination kit for detecting comma bacillus O139. The sequences of specific primers and a fluorescent probe in the kit are as follows: an upstream primer 5'-TCCTGAAATATTGTTGCGTTATAGG-3', a downstream primer 5'-ATCCCTATAGATGTAAGCACTTCA-3' and the fluorescent probe 5'-FAM-AAGAAAGATTTGAGATCATTTC-TAMRA-3', wherein FAM refers to a fluorescent reporter group, and TAMRA refers to a fluorescent quenching group. The fluoroscopic examination kit has the advantages of high bacterium detection sensitivity, good specificity, reduction of the false positive rate of general PCR amplification, and capability of realizing quick, accurate and special detection of the comma bacillus O139.

Description

(1) Technical field [0001] The invention relates to a fluorescent detection kit for Vibrio cholerae O139 and a detection method thereof. (2) Background technology [0002] Cholera is a severe infectious disease caused by Vibrio cholerae with diarrhea as the main symptom. In the past, it was believed that only the O1 type of Vibrio cholerae can cause disease, and the non-O1 type can only cause mild symptoms and cannot lead to large-scale epidemics. However, a cholera pandemic caused by a new type of non-O1 Vibrio cholerae, O139, broke out in India and Bangladesh in 1992. In recent years, large-scale epidemics caused by Vibrio cholerae O139 have also occurred in many areas in my country to varying degrees. Especially in recent years, the proportion of O139 cholera outbreaks is still rising. [0003] The traditional methods for detection of Vibrio cholerae mainly include morphology, biochemistry, coagulation test, etc. The cycle of these detections is long, generally more tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCY02A50/30
Inventor 张政金大智朱水荣
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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