Non-diagnostic method for detecting Vibrio vulnificus through TaqMan probe fluorescence RT-PCR

A technology of RT-PCR and Vibrio vulnificus, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. It can solve the problems of low specificity, achieve high sensitivity, and reduce contamination of amplification products. chance of reducing the effect of experimental steps

Inactive Publication Date: 2012-12-19
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

For example, Mark S. Campbell established a real-time PCR detection method based on SYBR Green based on the Vibrio vulnificus hemolysin gene (vvhA),

Method used

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  • Non-diagnostic method for detecting Vibrio vulnificus through TaqMan probe fluorescence RT-PCR
  • Non-diagnostic method for detecting Vibrio vulnificus through TaqMan probe fluorescence RT-PCR
  • Non-diagnostic method for detecting Vibrio vulnificus through TaqMan probe fluorescence RT-PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0104] Detection of Vibrio vulnificus in Artificial Stool Specimens

[0105] ① Take 1g of healthy human feces and put it in a sterile test tube;

[0106] ② Add 9ml of sterile distilled water and shake to make it homogenized;

[0107] ③ Aspirate 200 μl of feces with the tip of the pipette cut off, and extract DNA with a fecal DNA extraction kit as a negative control for subsequent experiments;

[0108] ④ Take 20 μl of the bacterial solution obtained above for each concentration gradient and add it to 180 μl of homogenized feces to prepare 8.0×10 Vibrio vulnificus. 6 CFU / ml-8CFU / ml artificially contaminated fecal samples with 7 concentration gradients, and 4 parallel samples for each concentration gradient;

[0109] ⑤ The first group of parallel samples directly extracted DNA with QIAamp DNA Stool Mini kit, and then performed real-time PCR and conventional PCR at the same time, using feces without bacterial solution as a negative control;

[0110] ⑥ Add 200 μl of artificially...

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Abstract

The invention discloses a non-diagnostic method for detecting Vibrio vulnificus through TaqMan probe fluorescence RT-PCR. The method comprises the following steps: comparing through adopting the DNA sequence of a Vibrio vulnificus hemolysin A (vvhA) gene as a target sequence, selecting a conserved region, determining the conserved sequence of the vvhA gene, and designing the real time PCR primers and the probes of the Vibrio vulnificus. The Vibrio vulnificus RT-PCR detection method which utilizes the specific fluorescence probes to implement a completely-closed tube type operation enables the amplified product pollution opportunity to be substantially reduced, the experiment steps, the experiment time and the reagent consumption to be reduced, the PCR in the invention to be different from routine PCR, electrophoretic identification to be not needed, and the appearance of a false positive result to be difficult because of difficult environmental protection. The method which can rapidly detect pathogenic bacteria has the advantages of high sensitivity, high specific degree and high lower limit of detection, can be applied to the detection of the Vibrio vulnificus in frontier port environment water, aquatic products and night soil of diarrhoea patients, and has a very high application value.

Description

technical field [0001] The invention relates to a non-diagnostic method for detecting Vibrio vulnificus by fluorescent RT-PCR with a TaqMan probe. Background technique [0002] Vibrio vulnificus (Vibrio vulnificus) is a Gram-negative halophilic Vibrio that widely exists in warm water environments, and is a common pathogen in estuaries and marine environments. Infecting healthy humans and animals mainly by contaminating seafood and infecting wounds, Vibrio vulnificus can cause sepsis and wound infections. In recent years, there have been reports of gastroenteritis caused by consumption of seafood contaminated by Vibrio vulnificus. The main symptoms are vomiting, diarrhea and abdominal pain. The case fatality rate of wound infection caused by Vibrio vulnificus is as high as 50%, and the case fatality rate of sepsis caused by Vibrio vulnificus infection is as high as 75%, especially in immunosuppressed patients and people with excessive iron loading in the body. In the monito...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/63
Inventor 韩辉徐宝梁李海山胡群刘丰周蕾
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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