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Isothermal amplification method of vibrio parahaemolyticus VP nucleic acid

A technology of vibrio hemolyticus and amplification reaction, which is applied in the direction of biochemical equipment and methods, DNA/RNA fragments, microbial determination/inspection, etc., and can solve the problem of inability to distinguish live bacteria from dead bacteria, etc.

Active Publication Date: 2014-02-12
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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  • Isothermal amplification method of vibrio parahaemolyticus VP nucleic acid
  • Isothermal amplification method of vibrio parahaemolyticus VP nucleic acid
  • Isothermal amplification method of vibrio parahaemolyticus VP nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Example 1. Design of special primers and probes for detection of Vibrio parahaemolyticus (VP) by real-time fluorescent nucleic acid constant temperature amplification

[0169] 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:

[0170] (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

[0174] Example 2 Preparation of a real-time fluorescent nucleic acid constant temperature amplification detection kit for Vibrio parahaemolyticus VP

[0175] 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.

[0176] Wherein, the capture probe sequence is as SEQ ID NO: 2;

[0177] T7 primer sequence such as SEQ ID NO: 3;

[0178] nT7 primer sequence such as SEQ ID NO: 4;

[0179] EV detection probe sequence such as SEQ ID NO: 5;

[0180] Internal standard detection probe sequence such as SEQ ID NO: 6;

[0181] Internal standard sequence such as SEQ ID NO: 7;

[0182] The capture probe exists...

Embodiment 3

[0215] Example 3 Sensitivity detection of pure bacteria

[0216] 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:

[0217] (1) Bacterial culture, counting and dilution

[0218] 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.

[0219] (2) Nucleic acid extraction

[0220] 2.1 Add 200μl of lysis solution (containing HEPES 35mM, (NH4)2SO4 20mM) and 200μl of sample solution to the sample processing tube (1.5mL centrifuge tube), and use the lysis solution to lyse the Vibrio parahaemolyticus (VP) in ...

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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 present invention involves the field of biological testing technology of pathogenic microorganisms in food source, and specificly involves the real -time fluorescent nucleic acid constant temperature amplification inspection of specific target capture technologies and real -time fluorescent nucleic acid constant temperature amplification detection technologyUse primers, probes and related kits. Background technique [0002] Vibrio Parahaemolyticus (VP) is a Gram -negative salt -love, which is one of the important pathogens that cause food -oriented diseases, which can cause patients with diarrhea, intestinal spasm, nausea, vomiting, fever, fever, fever, fever, fever, fever, fever, feverTypical gastroenteritis reaction.A variety of foods such as aquatic products and pickled products often occur in pollution.Data since 1998 showed that the scale of food poisoning caused by Hemorrhea and the exposure of crowds caused a significant increase in the scale of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
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