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Method for detecting suspension chip of multiple PCR products

A detection method and product technology, applied in the field of improvement of the conditions in the suspension chip detection method, can solve the problems of poor specificity, carcinogenicity, indistinguishable fragments of similar size, etc., and achieve the effect of flexible design

Inactive Publication Date: 2009-10-21
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For PCR products with similar or equal fragment lengths, agarose gel electrophoresis will be powerless
In addition, agarose gel electrophoresis uses ultraviolet light to excite fluorescent dyes to generate fluorescence to determine whether there are PCR products, and the detection sensitivity is relatively low
Moreover, agarose gel electrophoresis judges the product by the size of the fragment, which has poor specificity and cannot distinguish non-specifically amplified fragments of similar size
Moreover, commonly used dyes such as ethidium bromide are carcinogenic, and frequent operation is harmful to health.

Method used

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  • Method for detecting suspension chip of multiple PCR products
  • Method for detecting suspension chip of multiple PCR products
  • Method for detecting suspension chip of multiple PCR products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: The suspension chip detection method of PCR product is used for five kinds of bioterrorism factors Bacillus anthracis, Yersinia pestis, Brucella, Francisella tularensis, Burkholderia pseudomallei six-fold PCR product detection

[0046] 1. Determine the diagnostic fragments of the above five bacteria, select capB and an identification sequence on the chromosome for anthrax, select an identification sequence on the chromosome for plague, and select the specific fragment BCSP31 of the genus Brucella for Brucella, through NCBI's GeneBank Obtain candidate gene sequences, use software to design primers, and synthesize related sequences. The downstream primers are labeled with biotin at the 5' end, see Table 1, and these primers are diluted to 10 μmol / L.

[0047] Table 1 Primer Sequence

[0048]

[0049] 2. Multiplex PCR reaction using the above primers to amplify the above five bacteria to be detected.

[0050] 3. According to the nucleotide sequence in the a...

Embodiment 2

[0057] Example 2: Coupling of corresponding probes to microspheres

[0058] 1) Select coded microspheres with different numbers respectively, and oscillate the microsphere suspension with a vortex shaker to mix the microspheres evenly.

[0059] 2) Take the above microspheres about 1.25×10 6 Each was transferred to a centrifuge tube, centrifuged at 14000g for 3-5min, and the supernatant was carefully aspirated.

[0060] 3) Add 50ul of 0.1mol / L 2-(n-morpholino)ethanesulfonic acid solution, shake for 20-30s, and sonicate for 20-30s to resuspend the microspheres.

[0061] 4) Dilute the synthesized oligonucleotide probe to 0.1 mmol / L with distilled water.

[0062] 5) Add 1 ul of the diluted probe to the microsphere suspension, shake and mix.

[0063] 6) Add 2.5ul of freshly prepared 10mg / mL EDC solution to the mixture of microspheres and probes, shake and mix.

[0064] 7) Wrap the centrifuge tube with aluminum foil to avoid light, oscillate on a vortex shaker at 400-600 rpm, an...

Embodiment 3

[0070] Embodiment 3: Capture and detection of the PCR product to be detected by the capture probe

[0071] i. Take 3500 each of the coupled detection probes in the hybridization solution so that the total amount is 33 μl (calculate the corresponding addition amount according to the counting result of the microspheres)

[0072] ii. Add 5-17ul of the PCR products of the five bacterial mixed templates to each tube to make the final volume 50ul, and mix well by pipetting.

[0073] iii. Denature at 95°C for 10 minutes.

[0074] iv. Hybridization at the hybridization temperature for a certain period of time.

[0075] v. Transfer to a filter plate and filter to remove unbound PCR products.

[0076] vi. Add 75ul 4ng / ul SA-PE 1×TMAC solution to each well, incubate at room temperature in the dark for 10min, and filter to remove unbound SA-PE.

[0077] vii. Add 75ul 1×TMAC solution to each well and shake to resuspend the microspheres.

[0078] viii. Check on the machine after the rea...

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Abstract

The invention discloses a non-diagnostic method for detecting a suspension chip of PCR products. The chip mainly comprises coded microspheres, a biotinylated primer, a capture probe, and streptavidin-biotin-phycoerythrin, and the method comprises the following steps that: the capture probe is coupled with corresponding microsphere of each size respectively, a red laser excites classified fluorescent lights on aspherical substrate, and the types are determined according to different colors of the spherical substrate, wherein the biotinylated primer shows the a primer needs biotinylation labeling during PCR; the microspheres coupled with the probe can be specifically combined with a PCR product labeled by an amplified biotin; and the streptavidin-biotin-phycoerythrin is combined with a biotin on the PCR product captured on the microspheres, a green laser excites the phycoerythrin, and the number of the reported fluorescent molecules combined on the spherical substrate is measured and is used for indirectly determining the content of the PCR product combined on the spherical substrate.

Description

technical field [0001] The invention provides a suspension chip detection method for multiple PCR products, and also relates to the improvement of conditions in the suspension chip detection method. Background technique [0002] Polymerase chain reaction (PCR) is an in vitro amplification method that simulates natural DNA replication. The advent of PCR technology has brought a revolution to molecular diagnosis. PCR technology has become the basis of molecular diagnosis. The current PCR product detection method is agarose gel electrophoresis detection, and agarose gel can only distinguish DNA fragments with a difference of 100 bp. For PCR products with similar or equal fragment lengths, agarose gel electrophoresis will be powerless. In addition, agarose gel electrophoresis uses ultraviolet light to excite fluorescent dyes to generate fluorescence to determine whether there are PCR products, and the detection sensitivity is relatively low. Moreover, agarose gel electrophor...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 王静孙肖红杨宇文海燕刘衡川胡孔新
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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