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Nucleic acid membrane chromatographic fast detecting method and its test paper bar and use thereof

A detection method and technology of test strips, applied in biological testing, measuring devices, analysis materials, etc., can solve problems such as complicated operation, contamination of PCR products, long operation time, etc.

Inactive Publication Date: 2006-08-02
USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR technology also has limitations and shortcomings in clinical diagnosis, such as
[0005] 1. False positives due to laboratory contamination easily caused by PCR products;
[0006] 2. The detection of results after nucleic acid amplification (agarose gel electrophoresis or ELISA) is complicated, requires professional laboratories and equipment, and requires high professional skills for operators;
[0007] 3. Agarose gel electrophoresis can only determine the size of the amplified product, and its specificity is not high;
[0008] 4. The operation time is long, and the detection time after amplification is about 1-2 hours for agarose gel electrophoresis, and 2-3 hours for ELISA detection
However, this invention uses the whole double-stranded DNA as an antigen detection, and cannot distinguish different DNA sequences, so it is non-specific and cannot be used for specific detection, such as the diagnosis of infectious diseases.

Method used

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  • Nucleic acid membrane chromatographic fast detecting method and its test paper bar and use thereof
  • Nucleic acid membrane chromatographic fast detecting method and its test paper bar and use thereof
  • Nucleic acid membrane chromatographic fast detecting method and its test paper bar and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Processing and preparation of nucleic acid detection test strip materials

[0139] 1) Polyester film (connection pad) treatment:

[0140] a) Preparation of polyester film treatment solution:

[0141] Tris-HCl (PH9.0) 100mmol

[0142] Tween-20 0.5%

[0143] PVP 100 mg

[0144] Purified water to 10ml

[0145] Mix well and set aside;

[0146] b) spread the polyester film in a stainless steel basin, and pour the treatment solution until all the polyester film is submerged;

[0147] c) Soak overnight in a clean environment;

[0148] d) Take out the polyester film and put it in a 37°C oven overnight;

[0149] e) Take it out and keep it dry and airtight.

[0150] 2) Glass fiber (sample pad) treatment

[0151] a) Preparation of glass fiber treatment liquid:

[0152] Tris-HCl (pH9.0) 0.4 mol

[0153] Na 2 CO 3 0.2 mole

[0154] Tween-20 1%,

[0155] PVP 100mg

[0156] Purified water to 10ml

[0157] stir overnight;

[0158] b) Cut the glass fiber ...

Embodiment 2

[0184] Sensitivity of nucleic acid test strip detection method

[0185] The PCR amplification product is 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256, 1 / 512, 1 / 1024 and 1 / A proportional dilution of 2048. Take 5 μl each for agarose gel electrophoresis. The gel electrophoresis conditions are: 1X TBE buffer, voltage 100V, electrophoresis time 30 minutes. At the same time, 5 microliters were taken for nucleic acid test strip detection. After hybridizing 5 microliters of samples and probes in 100 microliters of hybridization solution (TBS, Tris-HCl, pH7.5, 10 millimolar, 150 millimolar sodium chloride), spot on the sample pad, and observe the results after 5 minutes.

[0186] The sensitivity comparison results of test strips and gel electrophoresis after nucleic acid amplification are shown in the attached Figure 5 in, by attached Figure 5 It can be seen from the results that compared with traditional gel electrophoresis, its sensitivity is increased by 100-200 times. ...

Embodiment 3

[0188] Using the single-probe method, the detection of the nucleic acid PCR amplification product of Neisseria gonorrhoeae:

[0189] ●PCR amplification:

[0190] Potassium chloride (500 mmol) 2 microliters

[0191] Tris-HCl (pH8.3, 100mM) 2 microliters

[0192] Magnesium chloride (20 mmol) 2 microliters

[0193] dNTP (2mM) 2μl

[0194] Forward primer (biotin-labeled, 2 μM) 2 μl

[0195] Reverse primer (2 μM) 2 μl

[0196] Neisseria gonorrhoeae 4000

[0197] Taq DNA polymerase 0.5 unit

[0198] Total 20 μl

[0199] 95°C for 30 seconds

[0200] 60°C 30 seconds

[0201] 72°C for 30 seconds

[0202] 35 cycles in total

[0203] ●Test strip detection:

[0204]PCR amplicon 8 μl

[0205] Reverse FITC-labeled probe (2 μM) 2 μl

[0206] TBS (Tris-HCl 10mM pH7.5, NaCl 150mM) 100 microliters

[0207] 95C, 5 minutes, after cooling to room temperature, point on the test strip for 5 minutes and observe the results.

[0208] The results of single-probe Neisseria gonorrhoeae PC...

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Abstract

The present invention discloses a single-probe detection method of specific nucleic acid sequence amplification product, also discloses a double-probe detection method of specific nucleic acid sequence amplification product, and also discloses a test paper strip for detecting nucleic acid amplification product and application of said test paper strip. The invented method uses the general antibody A and antibody B, so that the standard test paper strip made up by using the invented method can be used for detecting nucleic acid sequence amplification product from any source.

Description

technical field [0001] The present invention relates to the detection method of specific nucleic acid sequence amplification product, more specifically, relates to the method for rapidly detecting target nucleic acid sequence amplification product by nucleic acid thin film chromatography, and the present invention also relates to the method for rapidly detecting target nucleic acid sequence amplification product test strips and their use in the detection of infectious disease pathogens. Background technique [0002] The enzyme-linked (ELISA) reagent / colloidal gold (test strip) technology based on the immune reaction has developed quite maturely and has a considerable scale of market. This kind of product has entered the laboratory departments of various hospitals and even ordinary people's families because of its simple operation, low price, reliable and fast detection speed, etc., and has brought many conveniences to people's diagnosis of diseases. See, for example, Zhou Yo...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/544G01N21/78G01N33/52
Inventor 尤其敏胡林林源吉吴汀滢罗英
Owner USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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