Method and kit for rapidly detecting nucleic acid of staphylococcus aureus and methicillin-resistant staphylococcus aureus

A staphylococcal nucleic acid and methicillin-resistant technology, applied in the field of biotechnology, can solve problems such as unpublished and reliable solutions, and achieve the effects of short time-consuming, simple detection method and high sensitivity

Pending Publication Date: 2022-05-13
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on the above characteristics of Cas12a, it is expected to be applied to the construction of a rapid detection method for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, but there is no public and reliable solution yet

Method used

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  • Method and kit for rapidly detecting nucleic acid of staphylococcus aureus and methicillin-resistant staphylococcus aureus
  • Method and kit for rapidly detecting nucleic acid of staphylococcus aureus and methicillin-resistant staphylococcus aureus
  • Method and kit for rapidly detecting nucleic acid of staphylococcus aureus and methicillin-resistant staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Detection of artificially synthesized nuc1 gene and mecA gene using CRISPR-Cas12a system

[0069] 1-1. Artificial synthesis of nuc1 gene and mecA gene

[0070] In this example, the specific gene nucl of Staphylococcus aureus and the specific gene mecA of methicillin-resistant Staphylococcus aureus were synthesized by Sangon and cloned into the pUC57-kana vector. The sequence information of the nuc1 gene and the mecA gene are shown in the following tables, SEQ NO.1 and SEQ NO.2, respectively.

[0071]

[0072]

[0073] 1-2. crRNA preparation

[0074] Design crRNA targeting nuc1 gene and mecA gene: nuc1-crRNA and mecA-crRNA, and submit RNA fragments to be synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The sequence information is shown in the table below.

[0075]

[0076] 1-3. Preparation of ssDNA fluorescent probe

[0077] The specific sequence information of the ssDNA fluorescent probe is: 5'-6FAM-TTTATTT-3'-BHQ1, that is, ssDNA labeled wi...

Embodiment 2

[0082] Example 2 Using the CRISPR-Cas12a system to detect the nuc1 gene and mecA gene amplified by LAMP

[0083] 2-1. Primer preparation for constant temperature amplification of LAMP nucleic acid

[0084] According to the sequence information of nuc1 gene and mecA gene, the corresponding amplification primer set for nuc1 gene and mecA was designed and synthesized by Suzhou Synbio Biotechnology Co., Ltd. The sequence information of the primers is shown in the table below.

[0085]

[0086]

[0087] 2-2. Using LAMP nucleic acid constant temperature amplification technology to amplify nuc1 gene and mecA gene

[0088]A 25 μL reaction system was used, and the specific components and dosages were as follows. The Primer mix is ​​FIP / BIP (16 μM), F3 / B3 (2 μM) and FLP / BLP (4 μM). Add 2.5 μL Primer mix, 2.5 μL 10× Isothermo Buffer (Mg 2+ free), 2μL 100mM Mg 2+ , 3.5μL dNTP (10mM each), 3.5μL H 2 O, 10 μL sample and 1 μL Bst 3.0 DNA / RNA Polymerase (8 U / μL). All components we...

Embodiment 3

[0092] Example 3 Nucleic acid detection of different concentrations of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus using CRISPR-Cas12a system

[0093] 3-1. Rapid extraction of nucleic acid from Staphylococcus aureus and methicillin-resistant Staphylococcus aureus

[0094] Take 1mL bacterial solution, centrifuge at 8000g for 2 minutes, remove 900μL supernatant, add 900μL TE solution, mix well, 8000g, centrifuge for 2 minutes, remove 920μL supernatant, add 10μL 20mg / mL lysozyme and 10μL 10% Triton X-100 , mix well, and react at 37°C for 15 minutes. The resulting solution is the rapid nucleic acid extraction solution.

[0095] 3-2. Constant temperature amplification and detection of nucleic acid of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus

[0096] Using 10 μL of rapid nucleic acid extraction solution as an amplification template, nucleic acid constant temperature amplification was carried out according to the method 2-2 in ...

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Abstract

The invention discloses a method and a kit for rapidly detecting nucleic acid of staphylococcus aureus and methicillin-resistant staphylococcus aureus. The method comprises the following steps: 1) extracting nucleic acid of a sample to be detected; (2) nucleic acid isothermal amplification: carrying out loop-mediated nucleic acid isothermal amplification on nucleic acid of a specific nuc1 gene of staphylococcus aureus or a specific mecA gene of methicillin-resistant staphylococcus aureus; and (3) detecting an amplification product, namely detecting the amplified nuc1 gene or mecA gene by utilizing a CRISPR-Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a) system. The CRISPR-Cas12a fluorescent probe method is adopted for detecting nucleic acid of the staphylococcus aureus and the methicillin-resistant staphylococcus aureus for the first time, and the method has the advantages of being high in sensitivity, high in specificity, short in consumed time, high in throughput, independent of large-scale experimental equipment and the like; and the kit can be conveniently used for base-layer rapid detection of nucleic acid of staphylococcus aureus and methicillin-resistant staphylococcus aureus in base-layer experiments and clinical first-line detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a rapid nucleic acid detection method and kit for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. Background technique [0002] Staphylococcus aureus, also known as aureus or Staphylococcus, commonly lives on human skin and is found in the nasal passages of about 25-30% of adults. When Staphylococcus aureus colonizes the human epidermis, it neither harms the host nor causes clinical symptoms. However, when the host has a wound on the skin surface or undergoes surgery, or when the body's own immunity is low, the resident Staphylococcus aureus can cause infection. Staphylococci often cause localized skin infections such as folliculitis, furuncles, and pustules, and can also cause abscesses and spread to bone (osteomyelitis), lungs (staphylococcal pneumonia), blood (bacteremia or sepsis), heart ( Endocarditis, which can damage heart valves) and other organs. St...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11C12R1/445
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2521/327C12Q2525/151C12Q2525/161C12Q2563/107
Inventor 孙义祥陈弘毅宋一之林恺铖胡慧杰王敬开郄兴旺孙博书
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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