Hand-foot-and-mouth disease (HFMD) related virus parallel detection liquid-phase chip and preparation method and application thereof

A parallel detection and liquid phase chip technology, which is applied in the field of molecular biology technology and clinical detection, can solve the problems of inconvenience, tedious and time-consuming laboratory and clinical diagnosis, and achieve the effect of saving detection cost

Inactive Publication Date: 2010-01-20
SHANDONG CENT FOR DISEASE CONTROL & PREVENTION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] According to the characteristics of diversification of viruses related to hand, foot and mouth disease, the current ELISA, RT-PCR and real-time RT-PCR methods used in the detection of hand, foot and mouth disease can only detect one virus at a time. And the needs of clinical diagnosis, the problem to be solved in the present invention is to provide a kind of hand, foot and mouth disease related virus parallel detection liquid phase chip and its preparation method and application, not only can detect multiple viruses that cause hand, foot and mouth disease at the same time, but also detect quickly, Accurate, time-saving and labor-saving, conducive to the early detection, early diagnosis and early treatment of hand, foot and mouth disease

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Coupling of probes to known numbered microspheres

[0027] 1. Select No. 26, No. 36 and No. 46 carboxyl microspheres (Luminex Company) respectively, and oscillate the microsphere suspension with a vortex shaker for 20 seconds to make the microspheres mix evenly.

[0028] 2. Take about 2×10 carboxyl microspheres of each number above. 3 Transfer each to a centrifuge tube and centrifuge at ≥8000×g for 2 min to precipitate carboxylated microspheres.

[0029] 3. Remove the supernatant and add 100μl dH 2 O, use a vortex shaker to resuspend the microspheres for 20s, centrifuge at ≥8000×g for 2min, and precipitate the carboxylated microspheres.

[0030] 4. Remove the supernatant, add 80 μl, 100 mM, pH = 6.2 sodium dihydrogen phosphate salt solution, and resuspend the washed carboxy microspheres with a vortex shaker for 20 seconds.

[0031] 5. Add 10μl, 50mg / ml Sulfo-NHS (with dH 2 O dilution), oscillate gently with a vortex shaker.

[0032] 6. Add 10μl, 50mg / ml ...

Embodiment 2

[0043] Example 2: Application of the liquid chip for parallel detection of hand, foot and mouth disease-associated virus according to the present invention in clinical detection

[0044] The technical process of detecting HFMD-related viruses by using the parallel detection liquid chip of HFMD-related viruses according to the present invention:

[0045] 1. Extraction of viral nucleic acid: RNA can be extracted using commercial kits, such as using QIGEN ViralRNA mini Extraction Kit (CAT: 52904) to extract viral RNA from clinical specimens or virus isolation cultures;

[0046] (1) Add 560 μl of AVL buffer to a 1.5ml centrifuge tube;

[0047] (2) Add 140 μl of clinical specimen or virus isolation culture to this centrifuge tube;

[0048] (3) Place at room temperature for 10 minutes, then centrifuge briefly;

[0049] (4) Add 560 μl of absolute ethanol, mix thoroughly for at least 15 seconds, and centrifuge briefly;

[0050] (5) Carefully add about 630 μl of the mixed solution t...

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PUM

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Abstract

The invention discloses a hand-foot-and-mouth disease (HFMD) related virus parallel detection liquid-phase chip and a preparation method and an application thereof. The HFMD related virus parallel detection liquid-phase chip in the invention mainly comprises microballoons, specific probes, biotinylated universal primer and streptavidin-phycoerythrin, which is characterized in that microballoons in the chip are Number 26, Number 36 and Number 46 microballoons; the specific probes are aminated specific probes aiming at virus VP1 area of EV71, CoxA16 and Echo9; the universal primer is enterovirus universal primer marked by activated biotin. In the invention, different probes can combine with different target molecules, different detection reactions can be classified by using laser for detecting color numbers of sphere matrix after reaction, thus having the quantitative or qualitative characteristics and high throughput advantage, thereby saving time and power and detection cost greatly.

Description

(1) Technical field [0001] The invention relates to a liquid phase chip and its preparation method and application, in particular to a liquid phase chip for parallel detection of hand-foot-mouth disease-related viruses and its preparation method and application, belonging to the technical fields of molecular biology technology and clinical detection. (2) Background technology [0002] Hand-foot-mouth disease (HFMD), also known as exanthematous stomatitis, is caused by enterovirus infection, and its clinical manifestations are mainly characterized by fever and rashes or herpes on the hands, feet, and oral cavity. An acute common infectious disease. It is more common in children, and infants and young children under 3 years old are more likely to get sick. HFMD is highly contagious, with complex transmission routes and rapid transmission, which can cause a pandemic in a short period of time. [0003] The pathogens causing HFMD are mainly types 2, 4, 5, 7, 9, 10, and 16 of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 李忠张华宁吕慧王显军毕振强张国宁张凯宁汪朝晖李艳艳
Owner SHANDONG CENT FOR DISEASE CONTROL & PREVENTION
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