Polydispersion liquid drop digital nucleic acid detection method and application thereof

A detection method and droplet technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of increasing the difficulty of experimental operation, limiting application, and long time consumption, so as to achieve low equipment dependence and reduce experimental cost , to avoid the effect of cutting

Pending Publication Date: 2022-07-15
SUN YAT SEN UNIV
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  • Application Information

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Problems solved by technology

However, in actual use, although the CRISPR-based method is used in combination with nucleic acid amplification technology to achieve high-sensitivity detection, due to the difference in reaction temperature and buffer component concentration, this combination is often divided into multiple stages, which cannot be achieved. One-step detection not only increases the difficulty of experimental operation, but also increases the po

Method used

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  • Polydispersion liquid drop digital nucleic acid detection method and application thereof
  • Polydispersion liquid drop digital nucleic acid detection method and application thereof
  • Polydispersion liquid drop digital nucleic acid detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Detection method of isothermal amplification nucleic acid based on polydisperse droplets

[0079] In this embodiment, the target molecule is detected using a polydisperse droplet-based isothermal amplification nucleic acid detection method.

[0080] The specific detection steps are as follows:

[0081] 1) A total of 3 μL of the LAMP reaction system containing the target molecule was transferred to an EP tube, and an appropriate amount of oil phase solution was quickly added to the EP tube to cover.

[0082] Wherein, in this embodiment, the target molecule is specifically the pEF-nsp125-3HA plasmid containing the new coronavirus N gene DNA (the construction method of the plasmid is carried out with reference to the conventional technical manual in the field, wherein the new coronavirus N gene DNA is obtained through the new coronavirus N Gene RNA (database No. NC-045512) was obtained by reverse transcription); the specific LAMP reaction system is shown in Tabl...

Embodiment 2

[0098] Example 2 CRISPR-Cas amplification-free nucleic acid detection method based on polydisperse droplets

[0099] In this example, the target molecule was detected using the polydisperse droplet-based CRISPR-Cas amplification-free nucleic acid detection method.

[0100] In this example, the CRISPR-Cas system used is selected as CRISPR-Cas12a. Of course, those skilled in the art can also choose other CRISPR-Cas systems to be substituted according to actual usage requirements.

[0101] The specific detection steps are as follows:

[0102] 1) After mixing the target molecule to be tested and the CRISPR-Cas12a detection reagent in the EP tube according to the composition shown in Table 2, quickly add an appropriate amount of oil phase solution to the EP tube to cover.

[0103] Among them, in this embodiment, the target molecule is specifically the DNA of the new crown N gene; the specific CRISPR-Cas reaction system is shown in Table 2.

[0104] Table 2

[0105] com...

Embodiment 3

[0112] Example 3 CRISPR-Cas amplification-free nucleic acid detection method based on polydisperse droplets

[0113] In this example, the target molecule was detected using the polydisperse droplet-based CRISPR-Cas amplification-free nucleic acid detection method.

[0114] In this embodiment, the CRISPR-Cas system used is selected as CRISPR-Cas13a. Of course, those skilled in the art can also choose other CRISPR-Cas systems to be substituted according to actual usage requirements. CRISPR-Cas systems useful in the present invention include, but are not limited to, CRISPR-Cas12a, CRISPR-Cas13a.

[0115] The specific detection steps are as follows:

[0116] 1) After mixing the target molecule to be tested and the CRISPR-Cas13a detection reagent in the EP tube according to the composition shown in Table 2, quickly add an appropriate amount of oil phase solution to the EP tube to cover.

[0117] Among them, in this embodiment, the target molecule is specifically the single-strand...

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Abstract

The invention discloses a polydispersion liquid drop digital nucleic acid detection method and application thereof, and the method comprises the following steps: mixing an oil phase solution, a detection sample and a detection reagent, generating liquid drops through a simple method, incubating, and quantitatively detecting target nucleic acid molecules in the sample according to the number and volume of positive liquid drops. According to the method, the reaction reagent is quickly divided into independent liquid drops through simple dispersion operation, so that the requirements on the environment and instruments are reduced, and meanwhile, the demand quantity of the sample and the detection reagent is remarkably reduced. Moreover, through rapid emulsion segmentation, nucleic acid amplification carried out before reagent segmentation or Cas protein cleavage of a target is effectively avoided, the target nucleic acid concentration is calculated based on the droplet size, and the detection sensitivity can reach an aM level.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a polydisperse droplet digital nucleic acid detection method and application thereof. Background technique [0002] Nucleic acid detection is an important means of infectious disease prevention, diagnosis and monitoring. In the related art, methods for nucleic acid detection include real-time quantitative polymerase chain reaction (qPCR) and nucleic acid isothermal amplification technology. Among them, real-time quantitative polymerase chain reaction (qPCR) is the gold standard for clinical nucleic acid detection and quantification. Especially with the development of microfluidic technology, digital PCR has gradually become a new generation of absolute quantitative PCR technology. In addition, nucleic acid isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2563/159C12Q2521/327C12Q2525/161
Inventor 王佳思薛莹莹王珂周建华
Owner SUN YAT SEN UNIV
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