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LAMP-based (loop-mediated isothermal amplification-based) visual fluorogenic and chromogenic genetic testing method for microorganisms

A gene detection and microbial technology, applied in biochemical equipment and methods, microbial measurement/inspection, fluorescence/phosphorescence, etc., can solve the problems of poor specificity, low sensitivity, time-consuming and laborious, etc., and achieve fast and repeatable detection Highly specific and specific effects

Inactive Publication Date: 2012-07-18
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional culture methods for detecting microorganisms require separation, screening, biochemical identification, and if necessary, serological identification, which generally takes 4 to 6 days, which is time-consuming and laborious, and has the disadvantages of low sensitivity, poor specificity, false positives, and time-consuming and laborious.
[0004] Various conventional PCR (Polymerase Chain Reaction polymerase chain reaction) methods have the advantages of strong sensitivity, high specificity, simplicity and speed, and there are many reports on microbial gene detection. Problems such as false positives and the need for special equipment limit its application in grassroots units

Method used

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  • LAMP-based (loop-mediated isothermal amplification-based) visual fluorogenic and chromogenic genetic testing method for microorganisms
  • LAMP-based (loop-mediated isothermal amplification-based) visual fluorogenic and chromogenic genetic testing method for microorganisms
  • LAMP-based (loop-mediated isothermal amplification-based) visual fluorogenic and chromogenic genetic testing method for microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: composition of reaction system

[0061] a) LAMP reaction solution

[0062] The LAMP reaction solution was composed of LAMP 10×buffer 2.5 μl, 20 μmol / L internal primers FIP and BIP each 2.0 μl, 20 μmol / L external primers F3 and B3 each 1.0 μl, 25 mmol / L dNTPs 3.0 μl, 50 mmol / L MgSO4 3.0 μl, 1.0 mol / L betaine 5 μl, Bst DNA polymerase large fragment 1.0 μl, sterile double distilled water 2.0 μl, the total volume of the reaction solution is 22.5 μl.

[0063] b) Fluorescent chromogen

[0064] The fluorescent chromogenic agent is composed of 1 μl of calcein 10μmol / L-20mmol / L and 60μmol / L-0.1mol / LMnCl 2 0.5 μl composition.

Embodiment 2

[0065] Example 2: Using this detection method to detect Salmonella in food

[0066] a. Food sample pretreatment

[0067] Weigh 25g (mL) of 3 kinds of food samples contaminated by Salmonella and 3 kinds of non-contaminated food samples, put them into 6 sterile homogenizing cups filled with 225mL BPW, homogenize at 8000r / min~10000r / min for 1min ~2min, or put it in a sterile homogeneous bag filled with 225mL BPW, and beat it with a slap-type homogenizer for 1min~2min; if the sample is liquid, it does not need to be homogenized, just shake and mix well; Transfer the sample to a 500mL Erlenmeyer flask. If a homogeneous bag is used, it can be cultured directly at 36°C±1°C for 4 hours; if it is a frozen product, it should be thawed at below 45°C for no more than 15 minutes.

[0068] b. Preparation of bacterial template DNA

[0069] Take 100 μL of bacterial culture, centrifuge at 12000r / min for 2min, remove the supernatant, add 50μL of sterile water, mix well, bathe in 100℃ water fo...

Embodiment 3

[0084] Example 3: Using this detection method to detect Shigella in food

[0085] a. Food sample pretreatment

[0086] Take 3 kinds of food samples contaminated by Shigella and 3 kinds of non-contaminated food samples, respectively weigh 25g (mL) samples into 6 sterile homogeneous cups filled with 225mL BPW, at 8000r / min~10000r / min Homogenize for 1 min to 2 min, or put it in a sterile homogenizing bag filled with 225mL BPW, and beat it with a beating homogenizer for 1 min to 2 min; if the sample is liquid, it does not need to be homogenized, just shake and mix; Aseptically transfer the sample to a 500mL Erlenmeyer flask. If a homogeneous bag is used, it can be cultured directly at 36°C±1°C for 4 hours; if it is a frozen product, it should be thawed at below 45°C for no more than 15 minutes.

[0087] b. Preparation of bacterial template DNA

[0088] Take 100 μL of bacterial culture, centrifuge at 12000r / min for 2min, remove the supernatant, add 50μL of sterile water, mix well...

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Abstract

The invention discloses an LAMP-based visual fluorogenic and chromogenic genetic testing method for microorganisms, which relates to a genetic testing method for microorganisms. The testing method includes the following steps: (1) buffered peptone water (BPW) is used for culturing a sample to be tested according to a national standard method for 4 hours; (2) a water boiling method is used for extracting DNA (deoxyribonucleic acid) from the sample to be tested; (3) the DNA is added into an LAMP reaction system, and the temperature of 65 DEG C is kept for 1 hour; (4) by means of comparison with a control group, the result of the sample to be tested is observed with naked eyes, and qualitative analysis is carried out on the sample. Compared with the conventional culture method, the genetic testing method has the advantages that: testing is rapid, only taking 5 hours, and plus the extraction of the sample DNA, testing takes less than 6 hours in total; the specificity is good, and the sensitivity is high; the steps are simple, and the repeatability is high; and a plurality of samples can be tested at the same time. The genetic testing method can qualitatively test microorganisms, and can take the place of the conventional culture method and the serological testing method which are used up to now.

Description

technical field [0001] The invention relates to a microorganism gene detection method, in particular to a LAMP-based microorganism visual fluorescence color gene detection method, which is suitable for the qualitative detection of microorganisms. Background technique [0002] Microorganisms are a large group of organisms including bacteria, viruses, fungi, and some small protozoa and microscopic algae. Microorganisms are tiny, but they are closely related to human life. They cover many beneficial and harmful species, and are widely involved in many fields such as health, food, medicine, industry and agriculture, and environmental protection. One of the most important impacts of microorganisms on humans is the prevalence of infectious diseases, and 50% of human diseases are caused by viruses. Food contamination is a serious threat to human health, and food-borne pathogens are pathogens that cause bacterial food poisoning. Therefore, microbial detection is of great significa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 王业富卢暄郑虎
Owner WUHAN UNIV
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