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Method for semi-quantitatively detecting pathogenic nucleic acid by adding internal control nucleic acid

An internal control nucleic acid and semi-quantitative technology, which is applied in the field of semi-quantitative detection of pathogenic nucleic acid by adding internal control nucleic acid, can solve the problems of false negatives and achieve the effects of convenient operation, accurate detection results and low production cost

Inactive Publication Date: 2014-01-01
HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] The purpose of the present invention is to provide a solution to the problem that any problem in the whole process of the existing nucleic acid diagnosis technology (from the extraction of the sample to the amplification of the target fragment, and then to the use of test strips for detection) is prone to false negatives. A method for semi-quantitative visual detection of sample nucleic acid using internal control nucleic acid fragments

Method used

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  • Method for semi-quantitatively detecting pathogenic nucleic acid by adding internal control nucleic acid
  • Method for semi-quantitatively detecting pathogenic nucleic acid by adding internal control nucleic acid
  • Method for semi-quantitatively detecting pathogenic nucleic acid by adding internal control nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 (adopt the test strip method that adds internal control, detect HBV virus in the blood)

[0048] my country is a high-incidence area of ​​hepatitis B virus infection in the world. At present, hundreds of millions of people in the country are carriers of hepatitis B virus, and there are about tens of millions of chronic hepatitis patients. Every year, the cost of direct treatment of viral hepatitis in my country is about tens of billions (excluding the cost of health care products, the indirect losses caused by affecting studies, employment and work, etc.). Efficient and accurate detection of HBV is crucial in its prevention and treatment.

[0049] The HBV genome is a circular double-stranded DNA with a full length of 3182 bp (about 3.2 kb). The length of the double strand is asymmetric, and the negative strand (long strand) is complementary to the viral mRNA. Only the 5' end of the positive strand is fixed, and its length can be 50%-100% of the negative s...

Embodiment 2

[0063] Embodiment 2 (investigate the semi-quantitative performance of the test strip method adding internal control)

[0064] experiment process:

[0065] 1) PCR amplification: Six different concentrations of plasmid templates (5×10 1 , 5×10 2 , 5×10 3 , 5×10 4 , 5×10 5 , 5×10 6 copies / μL) and a certain concentration of HBV internal control nucleic acid fragments (50copies / μL) were mixed as templates, and HBV sample amplification-specific primers and corresponding internal control primers were used for double amplification in a single tube. The HBV internal control nucleic acid fragment sequence, the HBV primer sequence, the corresponding internal control nucleic acid fragment primer sequence, the reaction system and the reaction procedure are the same as in Example 1.

[0066] 2) Test strip detection: the implementation steps are the same as above, see the results Figure 5 . Depend on Figure 5 It can be seen from the results that when the concentration of the viral...

Embodiment 3

[0069]

[0070]

[0071]

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Abstract

The invention belongs to the field of nucleic acid detection and discloses a method for semi-quantitatively detecting pathogenic nucleic acid by adding internal control nucleic acid. Corresponding internal control is added in the whole process of extracting and amplifying target nucleic acid and testing by using a test paper, so that the internal control and a target segment are parallelly operated, and the semi-quantitative detection is performed finally through color development and intensity contrast of three strips, namely a detection line, an internal control line and a quality control line on the test paper. In the method, in the whole process of processing the target nucleic acid, the corresponding internal control is taken as a positive contrast, and false negative results due to links such as extraction, amplification or sample application errors are avoided in the processing of detecting by using the test paper. Meanwhile, by comparing color development intensity of the internal control line and a sample line and introducing the semi-quantitative function on the basis of the qualitative function of the immunochromatographic test paper to estimate the copy number of tested samples, the detection results are more detailed, accurate and reliable. The method has the advantages of convenient and quick operation and capacity of meeting the actual clinical requirement.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and relates to a method for semi-quantitative detection of pathogenic nucleic acid by adding internal control nucleic acid, in particular to introducing an internal control similar to the nucleic acid sequence to be tested during the whole process of detection of pathogenic nucleic acid to monitor the detection process, It not only eliminates false negatives, but also can perform semi-quantitative nucleic acid detection method by comparing the color intensity of the internal control line and the detection line on the test strip. Background technique [0002] Colloidal gold immunochromatography (gold-immunochromatography assay, GICA) method is a rapid immunological assay method developed in the late 1980s, which is the product of the combination of colloidal gold labeling technology and immunochromatography technology. The basic principle is to use the diafiltration concentration and capillar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N33/543G01N33/558
Inventor 周国华黄欢杨贤
Owner HUADONG RES INST FOR MEDICINE & BIOTECHNICS
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