Reagent for liquefying phlegm and protecting nucleic acid

A nucleic acid protection and reagent technology, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of time-consuming, high viscosity of sputum samples, incomplete mixing of samples, etc., and achieve the effect of simple production process and simple operation

Active Publication Date: 2018-12-07
ZHEJIANG JFK BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The sputum sample has a high viscosity, conventional liquefaction treatment takes a long time and the sample is not thoroughly mixed. At the same time, the nucleic acid in the sputum sample is easily degraded. At present, there is a lack of low-cost, simple production p

Method used

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  • Reagent for liquefying phlegm and protecting nucleic acid
  • Reagent for liquefying phlegm and protecting nucleic acid
  • Reagent for liquefying phlegm and protecting nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] A reagent for liquefaction of sputum and nucleic acid protection. Weigh 2.5 g of guanidine hydrochloride + 0.5 g of cysteine ​​+ 2 g of polypropylene particles and mix evenly to prepare the reagent of this embodiment.

[0024] Comparative experiment on the effect of this reagent and the traditional method on the liquefaction of sputum

[0025] Step 1: Prepare 8-10 cases of visually viscous sputum samples collected from the hospital, mix these sputum samples thoroughly, and take 4 3ml samples respectively, numbered 1-4;

[0026] Step 2: Add the sputum liquefaction and nucleic acid protection reagent of the present embodiment to No. 1 sample; Add 6ml dithiothreitol solution (2%) to No. 2 sample; Add 6ml trypsin solution (2%) to No. 3 sample buffer system containing pH7.0); add 6ml of sodium hydroxide solution (0.1mol / L) to sample No. 4, shake and mix the above samples for 5 minutes;

[0027] Step 3: Use filter paper to filter the 4 liquefied samples obtained in the previou...

Embodiment 2

[0029] Example 2: Comparison of the protective effect of this reagent and traditional methods on sputum nucleic acid after liquefaction

[0030] Step 1: Prepare 4-6 sputum samples of lung cancer patients collected from the hospital, mix these sputum samples thoroughly, and take 4 2ml samples respectively, numbered 1-4;

[0031] Step 2: add the sputum liquefaction and nucleic acid protection reagent of embodiment 1 in No. 1 sample; Add 6ml dithiothreitol solution (2%) in No. 2 sample; Add 6ml trypsin solution (2%) in No. 3 sample buffer system containing pH7.0); add 6ml of sodium hydroxide solution (0.1mol / L) to sample No. 4, shake and mix the above samples for 5 minutes;

[0032] Step 3: Fluorescent quantitative PCR method to detect mRNA in the sample: use the "Telomerase Reverse Transcriptase Subunit (hTERT) mRNA Detection Kit" developed by Zhejiang Jinfukang Biotechnology Co., Ltd. - hTERT mRNA in sample no. 4. The test results are shown in the table below. Samples 1-2 are...

Embodiment 3

[0035] Example 3: Comparison of nucleic acid protection effects of different formulation combination reagents

[0036] Step 1: Prepare 15-20 sputum samples of lung cancer patients collected from the hospital, mix these sputum samples thoroughly, take 20 samples of 2ml respectively, and use 4 samples as a group to obtain 5 groups of samples in total;

[0037] Step 2: Configure the combined protective agent as shown in the table below:

[0038]

[0039]

[0040] Step 3: Add reagent 1-reagent 5 to the 5 groups of sputum samples respectively; place the sample tubes at 37°C for 36 hours, 72 hours, 120 hours, and 168 hours for testing;

[0041] Step 3: Use the Telomerase Reverse Transcriptase Subunit (hTERT) mRNA Detection Kit" (National Machinery Note 20173404247) to detect the samples obtained in step 3 according to the operation steps in the product manual. The experimental results are shown in the table below. The results show that , reagent 1-reagent 5 within the scope o...

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Abstract

The invention provides a reagent for simultaneously liquefying phlegm and effectively protecting nucleic acid. The reagent contains guanidine, a cysteine derivative and a macromolecule inert particle,wherein guanidine is one or more of guanidine hydrochloride, guanidinium isothiocyanate, guanidine sulfate and guanidine carbonate; the cysteine derivative is one or more of cysteine, acetylcysteine,homocysteine, methylcysteine and cysteine hydrochloride; and the macromolecule inert particle is one or more of a polypropylene particle, a polyethylene particle, a polyvinyl chloride particle and apolystyrene particle. The reagent is a mixed solid of powder and particles and is capable of well permeating into a phlegm sample, and the phlegm sample is adequately mixed, so that phlegm can be adequately liquefied; and meanwhile, the reagent is capable of protecting nucleic acid in the phlegm sample from being degraded at a normal temperature for a long time. The reagent contains simple components and is low in cost, and the production process is simple, and the reagent is easy to use and operate and very suitable for wide clinical use.

Description

technical field [0001] The method belongs to the technical field of sample processing and preservation for medical testing, and relates to a sputum liquefaction and nucleic acid protection reagent. The technical key lies in promoting sample liquefaction and protecting nucleic acid in the sample from degradation. Background technique [0002] Phlegm is the secretion of the human respiratory tract. It is pushed from the lungs to the respiratory tract through the movement of bronchial cilia and epithelial cilia. Finally, it is coughed out from the trachea through the normal cough reflex of a person. Normal people have very little sputum. A small amount of mucus secreted by moist airways. When people inhale harmful microorganisms such as irritating gas, dust, pathogenic bacteria, and viruses, inflammation may occur in the upper respiratory tract; or if lung diseases occur, such as bronchiectasis, lung abscess, lung cancer, etc., the secretion of the respiratory tract will increa...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/10
Inventor 陈燃曹文静刘松林
Owner ZHEJIANG JFK BIOLOGICAL TECH
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