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A kind of vibrio parahaemolyticus vp nucleic acid constant temperature amplification method

A constant temperature amplification technology for hemolytic vibrio, applied in biochemical equipment and methods, DNA/RNA fragments, and resistance to vector-borne diseases, etc., can solve the problem of inability to distinguish live bacteria from dead bacteria

Active Publication Date: 2019-04-05
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection targets of the above two methods are DNA, which cannot distinguish live bacteria from dead bacteria, and false positive results are prone to occur.

Method used

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  • A kind of vibrio parahaemolyticus vp nucleic acid constant temperature amplification method
  • A kind of vibrio parahaemolyticus vp nucleic acid constant temperature amplification method
  • A kind of vibrio parahaemolyticus vp nucleic acid constant temperature amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0161] Example 1. Design of special primers and probes for detection of Vibrio parahaemolyticus (VP) by real-time fluorescent nucleic acid constant temperature amplification

[0162] In the present invention, the highly conserved segment without secondary structure in the VP toxR gene is selected as the amplified target sequence region (its nucleotide sequence is shown in Sequence 1 in the Sequence Listing), and according to the principle of primer probe design, DNA ATAR, DNAman Software and artificially designed special primers and probe sequences for real-time fluorescent nucleic acid constant temperature amplification detection of Vibrio parahaemolyticus (VP), and obtained the following specific sequences:

[0163] (1) A capture probe (TCO, Target Capture Oligo) that can specifically bind to the target nucleic acid (VP RNA) sequence of Vibrio parahaemolyticus (VP) as shown in Sequence 1 in the sequence listing, the capture probe’s The nucleotide sequence is 5'-aucyuccagucuc...

Embodiment 2

[0167] Example 2 Preparation of a real-time fluorescent nucleic acid constant temperature amplification detection kit for Vibrio parahaemolyticus VP

[0168] Using the special primers and probes provided in Example 1, the real-time fluorescent nucleic acid constant temperature amplification detection kit for Vibrio parahaemolyticus VP of the present invention was obtained. The kit contains capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, VP detection probe, internal standard detection probe, internal standard, M-MLV reverse transcriptase and T7 RNA polymerase etc. point.

[0169] Wherein, the capture probe sequence is as SEQ ID NO: 2;

[0170] T7 primer sequence such as SEQ ID NO: 3;

[0171] nT7 primer sequence such as SEQ ID NO: 4;

[0172] EV detection probe sequence such as SEQ ID NO: 5;

[0173] Internal standard detection probe sequence such as SEQ ID NO: 6;

[0174] Internal standard sequence such as SEQ ID NO: 7;

[0175] The capture probe exists...

Embodiment 3

[0208] Example 3 Sensitivity detection of pure bacteria

[0209] With the kit of the present invention (see Example 2 for composition), the detection concentration is 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL of Vibrio parahaemolyticus (VP) in food samples, and set up a negative control. The specific method includes the following steps:

[0210] (1) Bacterial culture, counting and dilution

[0211] Take the standard strain of Vibrio parahaemolyticus and streak the alkaline peptone water (APW) plate, culture at 37°C for 12 hours, count by turbidimetry, and dilute with normal saline to quantify to 10 7 CFU / mL, and diluted to 10 times with normal saline 2 CFU / mL.

[0212] (2) Nucleic acid extraction

[0213] 2.1 Add 200 μl of lysate (containing HEPES 35mM, (NH4)2SO4 20mM) and 200μl of sample solution to the sample processing tube (1.5mL centrifuge tube), and use the lysate to lyse the Vibrio parahaemolyticus (VP) in the sample to be...

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Abstract

The invention discloses an isothermal amplification method of vibrio parahaemolyticus VP nucleic acid. The isothermal amplification method specifically comprises the following steps of: carrying out amplification in a reaction system, wherein the reaction system contains a specific primer pair for amplifying the vibrio parahaemolyticus VP. The method disclosed by the invention is high in specificity, high in sensitivity and low in pollution, avoids false positive performance and quickly amplifies a to-be-measured sample containing VPRNA (completing detection with 50 minutes normally), has characteristics of being high in detection efficiency and high in accuracy, and has wide application prospect.

Description

technical field [0001] The invention relates to the technical field of biological detection of food-borne pathogenic microorganisms, in particular to the real-time fluorescent nucleic acid constant temperature amplification detection of Vibrio parahaemolyticus (VP) which combines specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology Primers, probes and related kits used. Background technique [0002] Vibrio parahaemolyticus (Vibrio Parahaemolyticus, VP) is a Gram-negative halophilic bacillus, which is one of the important pathogens causing foodborne diseases, which can cause diarrhea, intestinal cramps, nausea, vomiting, fever, etc. Typical gastroenteritis reaction. Vibrio parahaemolyticus contamination often occurs in a variety of foods such as aquatic products and pickled products. Data since 1998 show that the scale of food poisoning caused by Vibrio parahaemolyticus and the scale of population ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/04C12Q1/6844C12Q2561/113C12Q2563/107Y02A50/30
Inventor 张长明于明辉尹华立朱凤居金良
Owner SHANGHAI RENDU BIOTECH