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Kit for quantitatively detecting vibrio parahaemolyticus in food and clinic sample

A technology for quantitative detection of hemolytic vibrio, applied in the field of kits, can solve the problems of delayed diagnosis and treatment, high fatality rate, and long time consumption, and achieve the effect of good sensitivity and rapid early diagnosis

Active Publication Date: 2010-05-19
XIAN TIANLONG SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional detection method of Vibrio parahaemolyticus mainly relies on the biological characteristics of Vibrio parahaemolyticus (such as Gram-negative bacteria, halophilic acid, pyrogenic hemolysin, etc.) combined with a variety of biochemical reagents for detection, This method has certain specificity and sensitivity, but the operation is cumbersome and time-consuming, and it is mainly used for the detection of large-scale samples
Due to the rapid progress of Vibrio parahaemolyticus infection, drug resistance, and high mortality rate, traditional detection methods may delay diagnosis and treatment. Therefore, it is necessary to develop early and rapid detection technology

Method used

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  • Kit for quantitatively detecting vibrio parahaemolyticus in food and clinic sample
  • Kit for quantitatively detecting vibrio parahaemolyticus in food and clinic sample
  • Kit for quantitatively detecting vibrio parahaemolyticus in food and clinic sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: the development of Vibrio parahaemolyticus detection reagent

[0056] 1. Design of primers and probes: through sequence comparison and analysis of all the existing Vibrio parahaemolyticus nucleic acid sequences in the Genbank database and the nucleic acid sequences reported in published literature at home and abroad, to identify pathogenic bacteria with Vibrio parahaemolyticus The relevant main virulence factor exotoxin gene is the amplified target site, selects a highly conserved segment without secondary structure, and uses software and manual design of multiple pairs of primers and probes according to the basic principles of primer and probe design.

[0057] 2. Selection of clinical samples: According to the relevant literature reports at home and abroad, the samples for testing can be selected from various food samples, such as seafood or salt pickled products. The common ones are crabs, squid, jellyfish, fish, yellow mud snails, etc. , followed by egg...

Embodiment 2

[0075] Embodiment 2: Vibrio parahaemolyticus detection kit and its application

[0076]1. Prepare a kit including the following components: 2 tubes of DNA extraction solution (500ul / tube), 20 tubes of PCR amplification reaction solution (20ul / tube), 1 tube of negative quality control (100ul / tube), positive quality control 1 tube of product (50ul / tube), 4 tubes of quantitative standard (50ul / tube).

[0077] 2. Specimen collection, transportation and storage

[0078] (1) Specimen collection: including various food specimens, such as blood, urine, sputum, pus, and puncture fluid, etc., as well as various clinical specimens from hospitals, such as vomit, blood, feces, etc., according to PCR Sampling is required to operate; operate in accordance with the national microbiological inspection standards for cosmetics. Sealed for inspection.

[0079] (2) Specimen storage and delivery: The specimens can be used for testing immediately, or can be stored at -20°C for testing, and the st...

Embodiment 3

[0088] Example 3: Quantitative detection of clinical samples using Vibrio parahaemolyticus detection kit

[0089] The clinical samples come from the semi-solid culture of Nanjing Children's Hospital, and 50 ul of the upper layer of the culture is directly taken as the test object. Specimen processing, DNA extraction, PCR reaction and result analysis are carried out with reference to Example 2.

[0090] After the PCR reaction, adjust the analysis parameters according to the amplification curve to make the standard curve under the standard curve (Std curve) window reach the best (ie, the absolute value of the correlation value > 0.97), and then analyze the clinical samples. The test results of clinical samples are attached Figure 5 Shown: The Ct values ​​of the amplification curves of the six clinically positive samples are 23.83, 24.63, 26.02, 27.05, 27.28, and 29.61, respectively. Combined with the obvious exponential growth period of the amplification curves, all of them can...

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Abstract

The invention discloses a kit for quantitatively detecting vibrio parahaemolyticus in food and clinic sample. The kit comprises respectively packaged DNA extracting solution, PCR amplification reaction liquid, negative standard substance, positive reference material, quantitative standard substance and a plurality of sealed reagent bottles or test tubes. The invention is characterized in that the PCR amplification reaction liquid contains forward primer VP-F, reverse primer VP-R, oligonucleotide probe VP-P, heat-resistant DNA polymerase and deoxyribonucleoside triphosphate for target polynucleotide amplification. The vibrio parahaemolyticus pathogen in food samples and samples of puke, blood and dejection of patient with clinic gastrointestinal inflammation or visceral hemorrhage can be detected, thereby being capable of providing reliable experimental evidences for sensitively and quickly diagnosing vibrio parahaemolyticus pollution and infection and repetitive infection of vibrio parahaemolyticus at an early time; and simultaneously, the amount can be accurately fixed, thereby being capable of effectively monitoring clinic drug administration.

Description

technical field [0001] The invention relates to a kit, in particular to a kit for quantitatively detecting Vibrio parahaemolyticus in food and clinical samples. The kit uses real-time fluorescence quantitative polymerase chain reaction technology to early and quickly judge whether a sample is parahaemolyticus. Hemolytic Vibrio contamination. Background technique [0002] Vibrio parahaemolyticus (also known as Vibrio Parahaemolyticus VP) is a Gram-negative polymorphic bacillus or Vibrio slightly curved. This bacterium is halophilic and acid-phobic, and cannot grow on salt-free medium. It reproduces rapidly in 3% to 6% saline, and every 8 to 9 minutes is a cycle, and it stops growing when it is lower than 0.5% or higher than 8% saline. It will die in 1-3 minutes in vinegar, inactivate in 5-10 minutes when heated to 56°C, and die in 5 minutes in 1% hydrochloric acid. It exists widely in nature and is an opportunistic pathogen that can cause human and animal diseases, visceral...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64C12R1/63
Inventor 彭年才李红东赤宏涛焦刚
Owner XIAN TIANLONG SCI & TECH
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