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Suspension chip-based multiple solid phase amplification detection method

A suspension chip and detection method technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of low amplification efficiency, limited application scope, etc. Wide application prospects and the effect of improving reaction efficiency

Inactive Publication Date: 2010-12-08
SUZHOU INST OF NANO TECH & NANO BIONICS CHINESE ACEDEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the fact that the traditional solid-phase bridge multiplex PCR is a solid-liquid phase reaction, its amplification efficiency is significantly lower than that of liquid-phase PCR, which limits its application range.

Method used

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  • Suspension chip-based multiple solid phase amplification detection method
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  • Suspension chip-based multiple solid phase amplification detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The comparison of embodiment 1 nucleic acid immobilization method

[0029] Nucleic acids are immobilized using two methods

[0030](1) Use 3-aminopropyltrimethoxysilane to aminosilanize the surface of the microchip, and use glutaraldehyde as the "arm molecule" to connect the aminated microchip to the 5' amino-modified nucleic acid primer to immobilize the nucleic acid Primers, and unreacted aldehyde groups are blocked by sodium borohydride, etc.

[0031] The specific experimental steps are as follows:

[0032] 1. Shake the microspheres or microchips sufficiently, centrifuge at 10000r / min for 1min, discard the supernatant until the suspension of microspheres is neutral, and store in pure water.

[0033] 2. Add 250 μl of 6% APTES (solvent: 95% ethanol), oscillate sufficiently for 30 s, oscillate for 30 min at 10,000 r / min, centrifuge for 1 min, discard the supernatant, and add absolute ethanol to 1 ml. After shaking for 30s, centrifuge at 10000r / min for 1min, discard t...

Embodiment 2

[0051] Embodiment 2 Staphylococcus aureus multiplex solid-phase PCR detection

[0052] (1) Detected strains include ATCC25923, TCC6538, CMCC26001, 5H048, 05I073, 05K037, 05N074, 05L198, 05B008, 05B012, 05B033, 05B038, 05B041, 05B059, 05I056;

[0053] (2) All bacterial strains were cultured in constant temperature LB broth medium at 37°C for 20 hours to extract DNA, and the DNA was extracted using the CTAB method;

[0054] (3) Search for the sequence of Staphylococcus aureus through NCBI and published literature, and design specific primers for detection through Primer Premier5.0. The primer sequences are shown in Table 1, and the primers are diluted to 100 μM;

[0055] Table 1 Amplification primers for detection of Staphylococcus aureus

[0056]

[0057] (4) with SiO 2 The microchip is used as a solid-phase multiplex PCR chip, and a number of squares are produced on its surface through semiconductor micromachining technology as codes. The arrangement of the squares on the...

Embodiment 3

[0065] The multiple solid-phase RT-PCR detection of embodiment 3 human parainfluenza virus

[0066] (1) Detection of viruses HPIV-1, HPIV-2, HPIV-3 and HPIV-4. The virus strain was recovered at room temperature, and 200 μL of the virus liquid was inoculated in a culture plate with 70% growth of LLC-MK2 (Rhesus monkey kidney cells) cells. Observe the cytopathic changes every day until CPE (cytopethic effect) appears, harvest the culture medium and cells at -70°C for later use;

[0067] (2) Total viral RNA from infected cells was extracted with QIAamp Kit from QIAGEN, Germany, and the extraction method is detailed in the instruction manual. 200 μl of cell culture medium was extracted, and the final product RNA was eluted with 50 μl of DEPC-treated water, and the eluate was stored at -70°C for later use;

[0068] (3) The reverse transcription of total RNA into cDNA was completed through the M-MLV RTasecDNASynthesis Kit of Japan TAKARA Company. For the reverse transcription meth...

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Abstract

The invention relates to the field of molecule detection, in particular to a suspension chip-based multiple solid phase amplification detection method. The method of the invention comprises the following steps of: 1) preparing chips with different codes; 2) modifying the surfaces of the chips and fixedly connecting each pair of specific primers to a chip with a specific code respectively; 3) preparing DNA or RNA of a plurality of samples to be detected respectively; 4) performing multiple solid phase amplification; and 5) detecting amplified products by using fluorescent. The method can detect a plurality of target segments at the same time and effectively avoid the interference among primers and has high repeatability, specificity and sensitivity; and the whole method is easy to operate, requires no professional and has a wide application prospect in actual use.

Description

technical field [0001] The invention relates to the field of molecular detection, in particular, the invention relates to a suspension chip-based multiple solid-phase amplification detection method. Background technique [0002] In the past 10 years, with the rapid development of modern molecular biology, more reliable nucleic acid detection methods have been provided for clinical laboratory diagnosis, among which two technologies, nucleic acid molecular hybridization and nucleic acid amplification, are mainly used. The typical representative of nucleic acid amplification technology is the polymerase chain reaction (polymerase chain reaction, PCR), as the basis of molecular differential diagnosis (MDD), this technology plays an important role in the early identification of pathogens due to its high efficiency and rapidity. effect. And the technology is constantly improving and innovating in order to better meet the needs of accurate and efficient molecular differential diag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 李炯曹榕吕卓璇段德民鲍芳姜丽
Owner SUZHOU INST OF NANO TECH & NANO BIONICS CHINESE ACEDEMY OF SCI
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