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Kit and detection method for rapid detection of nucleic acid of mycoplasma pneumonia on basis of CRISPR/Cas12a

A technology of mycoplasma pneumoniae and detection method, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, can solve problems such as high price, and achieve simple detection methods, high-sensitivity visual detection, and convenience. The effect of quick result interpretation

Inactive Publication Date: 2020-05-22
国家卫生健康委科学技术研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used molecular diagnostic method for nucleic acid detection is PCR technology. Primers are designed for the ATPase gene sequence, P1 adhesin gene sequence, and the conserved region of 16SrRNA of Mycoplasma pneumoniae for PCR amplification. Real-time quantitative PCR can also be used for the detection of Mycoplasma pneumoniae Compared with ordinary PCR, it can shorten the sample turnaround time and reduce steps such as electrophoresis, but it is expensive and requires special equipment and technology, which limits its further popularization

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  • Kit and detection method for rapid detection of nucleic acid of mycoplasma pneumonia on basis of CRISPR/Cas12a
  • Kit and detection method for rapid detection of nucleic acid of mycoplasma pneumonia on basis of CRISPR/Cas12a
  • Kit and detection method for rapid detection of nucleic acid of mycoplasma pneumonia on basis of CRISPR/Cas12a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Rapid and sensitive detection of Mycoplasma pneumoniae nucleic acid fragments

[0040] 1.1 Nucleic acid preparation

[0041] The MP gene fragment in this case refers to the P1 gene fragment of MP in the NCBI database. The 399bp SEQ NO.1 of the P1 part gene was synthesized by Nanjing GenScript and constructed into the pUC57 vector, named pUC57-MP-P1.

[0042] Using the RPA amplification primers RPA-F1 SEQ NO.2 and RPA-R1 SEQ NO.3 of the present invention, referring to the RPA isothermal amplification operation steps, amplified to obtain the sample to be detected. The specific operation is as follows:

[0043] 1μL (10ng) pUC57-MP-P1 sample was subjected to RPA amplification reaction: 25μL 2*Buffer, 2μL RPA-F1, 2μL RPA-R1 and 2.5μL magnesium acetate, mixed well, reacted at 37°C for 20min, obtained the sample for The next step is nucleic acid testing.

[0044] 1.2 Design and preparation of MP-specific crRNA

[0045] Such as figure 2 As shown, the preparatio...

Embodiment 2

[0060] Embodiment 2: CRISPR / Cas12a detects Mycoplasma pneumoniae nucleic acid sensitivity

[0061] In the case of sensitivity detection, the pUC57-MP-P1 plasmid was converted to copy number according to the molecular weight, and a 10-fold serial dilution was performed to obtain 2*e7, 2*e6, 2*e5, 2*e4, 2*e3 per microliter , 2*e2, 2*e1 and 2*e0 copy number (copy / μL). 1 μL of gradient dilution samples were subjected to RPA amplification reaction: 25 μL 2*Buffer, 2 μL RPA-F1, 2 μL RPA-R1 and 2.5 μL magnesium acetate, mixed well, reacted at 37°C for 20 minutes, and obtained samples for the next step of nucleic acid detection.

[0062] According to the results obtained in Example 1, crRNA has a high sensitivity for detecting the P1 gene of Mycoplasma pneumoniae, so crRNA1 is used for subsequent detection. This test uses a 20 μL system as shown in Table 3, but is not limited to this, including the adjustment of the proportion of the corresponding components:

[0063] Table 3. Mycop...

Embodiment 3

[0069] Example 3: Rapid detection of Mycoplasma pneumoniae nucleic acid on clinical sample nucleic acid

[0070] In this example, the rapid detection of nucleic acid in clinical samples is performed. All samples and operations are completed in the laboratory. In this case, the sample is DNA obtained from oral swabs for CRISPR / Cas12a detection. In this example, a rapid nucleic acid release agent purchased from Novizyme was used to obtain pretreated nucleic acid. The steps are as follows: wash the oral swab with PBS and centrifuge, take 2 μL of centrifuged supernatant and add 20 μL of nucleic acid lysate, let it stand at room temperature for 3 minutes, add 20 μL of neutralizing solution, mix well and proceed to the next step of detection.

[0071] Using the RPA amplification primers SEQ NO.2 and SEQ NO.3 in the present invention, referring to the RPA isothermal amplification operation steps, take 2 μl of each sample to be tested for RPA pre-amplification to obtain the sample to ...

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Abstract

The invention discloses a CRISPR / Cas12a kit and a detection method for rapid detection of nucleic acid of mycoplasma pneumoniae. The CRISPR / Cas12a kit comprises a CRISPR / Cas12a detection system, wherein the CRISPR / Cas12a detection system comprises specific crRNA aiming at mycoplasma pneumoniae, a CRISPR / Cas12a protein and a single-stranded DNA reporting system, wherein the specific crRNA is any one or more designed and synthesized according to mycoplasma pneumoniae dsDNA sites, and the single-stranded DNA reporting system comprises am ssDNA FQ reporter used for fluorescence detection of a microplate reader and / or an ssDNA DB reporter used for immunogold test strip detection. According to the invention, CRISPR / Cas12a is firstly adopted to detect the nucleic acid sequence of mycoplasma pneumoniae, and has the advantages of high sensitivity, strong specificity, short time consumption, no dependence on large-scale experimental equipment and the like.

Description

technical field [0001] The invention relates to the field of gene detection of Mycoplasma pneumoniae, more specifically, to a method for rapidly detecting Mycoplasma pneumoniae nucleic acid based on CRISPR / Cas12a and a kit thereof, belonging to the field of biotechnology. Background technique [0002] Mycoplasma pneumoniae (MP) is a common pathogenic microorganism that mainly causes respiratory tract infections in humans, especially in children and adolescents. Mycoplasma pneumoniae infection is generally sporadic and widely present in all parts of the world. Its symptoms are generally general respiratory symptoms such as headache, sore throat, fever and cough. At present, the widely accepted pathogenic mechanism is MP through adhesion and cytotoxic effect. It can cause direct damage to respiratory epithelial cells, and can also cause severe pneumonia and other systemic damage through the immune system. [0003] Mycoplasma pneumoniae MP belongs to the Mollusca class and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804C12Q1/689C12Q1/04C12R1/35
CPCC12Q1/6804C12Q1/689C12Q2521/327C12Q2563/131C12Q2525/161C12Q2565/625C12Q2563/107
Inventor 马旭王鑫杰金孝华张璐
Owner 国家卫生健康委科学技术研究所
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