Sample pretreatment process suitable for nucleic acid detection test strip for aquatic diseases

A technology for sample pretreatment and detection of test strips, applied in the biological field, can solve the problems that samples cannot be used, nucleic acid test strips are false negative, and nucleic acid detection test strips cannot be popularized and applied, so as to improve the detection effect, simplify nucleic acid extraction and Effects of purification steps

Pending Publication Date: 2019-07-19
LUDONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, nucleic acid test strips are often troubled by false negatives during use, resulting in misjudgment
This is because many nucleic acid test strips have relatively high requirements for test samples, and nucleic acid samples with higher purity and concentration are required to obtain the expected test results. Therefore, the samples cannot be subjected to simple pretreatment, and often undergo sample lysis, Complex sample pretreatment processes such as extraction, precipitation, rinsing, drying, and redissolution, some of which have to go through the signal amplification stage, limit their practical application, which also makes nucleic acid detection test strips unable to be used in aquaculture. Important reasons for the promotion and application of basic units

Method used

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  • Sample pretreatment process suitable for nucleic acid detection test strip for aquatic diseases
  • Sample pretreatment process suitable for nucleic acid detection test strip for aquatic diseases
  • Sample pretreatment process suitable for nucleic acid detection test strip for aquatic diseases

Examples

Experimental program
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Effect test

Embodiment 1

[0043] (1) Streak inoculate Vibrio splendidus on 2216E solid medium plate and culture at 28°C for 24 hours, pick a single colony in 2216E liquid medium, culture overnight at 28°C with 200rpm shaking, and dilute the culture solution with normal saline to Concentrations were 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 CFU / mL, take 1 mL of bacterial culture solution, centrifuge at 8000 r / min for 5 min, discard the supernatant, add lysate (50 mM Tris-HCl, 10 mM EDTA, 10 mM NaCl, 1% SDS, 0.1 mg / L proteinase K) 1 mL, in a 70°C water bath for 10 min.

[0044] (2) Centrifuge the above treatment solution at 12,000 rpm for 5 minutes, take 800 μL of the supernatant and add it to a type A nucleic acid adsorption column, and place it for 2 minutes. Centrifuge at 12000 rpm for 1 min, discard the liquid in the collection sleeve, and repeat this step once;

[0045] (3) Add 1 mL of 80% ethanol (pH 5.5), and centrifuge at 12000 rpm for 1 min;

[0046] (4) Leave it open for 3 minute...

Embodiment 2

[0050] (1) Cut 0.5 g of the body wall tissue of sea cucumber into pieces, transfer it to a glass homogenizer, add 5 mL of homogenate solution (100 mM Tris-HCl, 500 mM EDTA, 20 mM NaCl, 10% SDS), and homogenize Transfer the ground homogenate to a centrifuge tube, add 20 μL of proteinase K (20mg / mL), invert and mix, place in a constant temperature water bath at 70°C for 20 minutes, and cool on ice.

[0051] (2) Centrifuge the above treatment solution at 10,000 rpm for 5 minutes, take 2.5 mL of the supernatant and add it to a B-type nucleic acid adsorption column, and place it for 5 minutes. Centrifuge at 10,000 rpm for 3 minutes, discard the liquid in the collection sleeve, and repeat this step once.

[0052] (3) Add 2.5 mL of 80% ethanol (pH 5.5), centrifuge at 12000 rpm for 1 min, and repeat this step once.

[0053] (4) Leave it open for 3 minutes, add 20 μL of deionized water to the center of the silica gel adsorption membrane, leave it for 2 minutes, centrifuge at 12000 rpm...

Embodiment 3

[0057] (1) Take 100 mL of aquaculture water samples, put them in a 100°C water bath for 10 min, and cool them down at room temperature.

[0058] (2) Centrifuge the above-mentioned treatment solution at 8500rpm for 5 minutes, take 17 mL of the supernatant and add it to a C-type nucleic acid adsorption column, and place it for 5 minutes. Centrifuge at 8500 rpm for 3 minutes, discard the liquid in the collection sleeve, and repeat this step once.

[0059] (3) Add 10 mL of 80% ethanol (pH 5.5), and centrifuge at 10,000 rpm for 1 min.

[0060] (4) Leave it open for 3 minutes, add 30 μL of deionized water to the center of the silica gel adsorption membrane, let it stand for 2 minutes, centrifuge at 12000 rpm for 1 minute, and use the obtained elution solution for subsequent detection tests.

[0061] (5) Take 50 μL of the above-mentioned eluent and add dropwise on the sample pad of the Vibrio resplendent nucleic acid test strip, place the test strip in a wet box, incubate at 37°C fo...

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Abstract

The invention relates to a sample pretreatment process suitable for a nucleic acid detection test strip for aquatic diseases, and belongs to the field of biotechnology. The process comprises the following steps: (1) taking different methods for different samples to perform cell disruption, and releasing nucleic acid to obtain a treatment liquid containing nucleic acid; (2) adsorbing the nucleic acid released by the step (1) through nucleic acid adsorption columns to enable the nucleic acid to attach to and gather on nucleic acid adsorption membranes of the nucleic acid adsorption columns; (3)removing impurities on the nucleic acid adsorption membranes; (4) eluting the nucleic acid on the nucleic acid adsorption membranes to obtain a nucleic acid eluent; and (5) dropwise adding the nucleicacid eluent to the nucleic acid test strip for testing. The process can greatly simplify the steps of nucleic acid extraction and purification, further improve the detection effect of the nucleic acid test strip, and improve the detection limit and the detection success rate.

Description

technical field [0001] The invention relates to a sample pretreatment process suitable for nucleic acid detection test strips of aquatic diseases, belonging to the field of biotechnology. Background technique [0002] Since the 1980s, the scale and output of my country's aquaculture industry have grown rapidly, and it has become one of the four pillar industries of my country's agriculture (grain, meat, aquatic products and poultry eggs). However, in recent years, with the increase in the scale of aquaculture and the increase in the intensity of aquaculture, the aquaculture environment has continued to deteriorate, and various aquaculture diseases have occurred frequently, causing huge economic losses to the aquaculture industry and seriously hindering the development of the aquaculture industry. sustainable development. According to preliminary statistics, there are currently 400-500 kinds of diseases that endanger aquaculture organisms. Among them, aquatic pathogenic micr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6834C12Q1/689C12Q1/04C12R1/63
CPCC12Q1/6806C12Q1/6834C12Q1/689C12Q2523/308C12Q2565/625
Inventor 张力支王磊王宏新石雪燕吴桂芬李玉洁肖雨晴马新冉吴红艳
Owner LUDONG UNIVERSITY
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