Nucleic acid parallel testing liquid-phase chip as well as 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 waste of drug resources, high detection costs, bacterial drug resistance, etc., and achieve the effect of saving detection costs

Active Publication Date: 2015-01-07
JINAN KAICHEN BIOTEC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This not only leads to the waste of drug resources, but also leads to the abuse of antibiotics, which leads to bacterial resistance; in addition, different bacterial infections require different antibiotic types, doses, and days. A basic principle of medication
[0004] At present, the commonly used methods for laboratory diagnosis of respiratory tract infection are pathogen culture method, ELISA (enzyme-linked immunosorbent assay), molecular biology reverse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR (rRT-PCR) etc. These methods can only detect one pathogen at a time. In the actual application process, the detection is cumbersome and requires a relatively large amount of reagents and clinical specimens, and the detection cost is high. Therefore, if new technologies can be used to detect these pathogens at the same time, it will be Provides great convenience for the diagnosis and treatment of this type of disease

Method used

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  • Nucleic acid parallel testing liquid-phase chip as well as preparation method and application thereof
  • Nucleic acid parallel testing liquid-phase chip as well as preparation method and application thereof
  • Nucleic acid parallel testing liquid-phase chip as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Coupling of probes to known numbered microspheres

[0060] (1) Select 11 kinds of different fluorescent colors (microspheres 11, 15, 22, 28, 36, 42, 49, 55, 56, 71, and 75) carboxylated microspheres (Luminex company), and oscillate with a vortex shaker Microsphere suspension, the time is 20s, so that the microspheres are mixed evenly.

[0061] (2) Take about 2×10 carboxyl microspheres of the above numbers respectively. 3 Transfer each to a centrifuge tube and centrifuge at ≥8000×g for 2 min to precipitate carboxylated microspheres.

[0062] (3) Remove the supernatant, add 100 μ ldH 2 O, use a vortex shaker to resuspend the microspheres for 20s, centrifuge at ≥8000×g for 2min, and precipitate the carboxylated microspheres.

[0063] (4) Remove the supernatant, add 80 μl, 100 mM, pH = 6.2 sodium dihydrogen phosphate salt solution, and resuspend the washed carboxyl microspheres with a vortex shaker for 20 s.

[0064] (5) Add 10μl, 50mg / ml Sulfo-NHS (with dH ...

Embodiment 2

[0076] Example 2: The application of the parallel detection liquid phase chip for respiratory tract infection pathogens of the present invention in clinical detection

[0077] The technical process of using the liquid chip for parallel detection of respiratory tract infection pathogens of the present invention to detect respiratory tract infection pathogens:

[0078] 1. Extraction of pathogen nucleic acid: RNA can be extracted using commercial kits, such as QIGEN Viral RNA mini Extraction Kit (CAT: 52904) to extract pathogen RNA from clinical specimens or pathogen isolation cultures; a total of 40 specimen nucleic acids were extracted, Of these, 30 were positive samples with known subtypes, 5 were negative controls, and 5 were samples of unrelated pathogens. According to the experimental requirements, another blank control was set up. Specimen information is as follows:

[0079]

[0080] (1) Add 560 μl of AVL buffer to a 1.5 ml centrifuge tube;

[0081] (2) Add 140 μl cl...

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Abstract

The invention belongs to the technical field of molecular biology and clinical test, and particularly discloses a nucleic acid parallel testing liquid-phase chip for respiratory tract infection pathogen diagnosis as well as a preparation method and application thereof. The nucleic acid parallel testing liquid-phase chip mainly comprises microspheres, probes, specific primers, biotinylated universal primers and streptavidin-phycoerythrin corresponding to respiratory tract infection relevant pathogens, and is characterized in that the microspheres are respectively coupled with the specific primers for 11 respiratory tract infection relevant pathogens to form microspheres No.11, No.15, No.22, No.28, No.36, No.42, No.49, No.55, No.56, No.71 and No.75; and the probes are aminated specific probes for 11 pathogen target genes. According to the nucleic acid parallel testing liquid-phase chip, the preparation method and the application, various respiratory tract infection relevant pathogens are parallelly detected, and 11 pathogens can be detected at the same time, so that great convenience is provided for laboratories and clinical tests of different diseases.

Description

(1) Technical field [0001] The invention belongs to the field of molecular biology technology and clinical detection technology, and in particular relates to a nucleic acid parallel detection liquid chip for diagnosis of pathogens of respiratory tract infection, a preparation method and application thereof. (2) Background technology [0002] Respiratory tract infection is a common infectious disease. Common causes are pathogens, and a few are caused by bacteria. Patients are highly contagious regardless of age, gender, occupation, and region. If not treated in time, the inflammation may spread to other organs and cause corresponding symptoms, and systemic symptoms will also aggravate. Common complications include sinusitis, otitis media, conjunctivitis, etc.; in young and frail children, upper respiratory tract infection tends to develop downward, causing bronchitis and pneumonia; Cause systemic and other complications such as sepsis, meningitis and nephritis. [0003...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C40B50/06
CPCY02A50/30
Inventor 李艳艳张凯宁
Owner JINAN KAICHEN BIOTEC
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