CRISPR detection primer group for mycoplasma pneumoniae and application of CRISPR detection primer group

A technology for detection of mycoplasma pneumoniae and primers, applied in the direction of microbe-based methods, microbiological measurement/testing, microbiology, etc., can solve the problem of inability to improve sensitivity, and achieve the goal of getting rid of the dependence on complex temperature-variable amplification instruments, high sensitivity, and broad The effect of applying the foreground

Active Publication Date: 2020-02-18
广州微远医疗器械有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in principle, the essence of its single-stage amplification reaction has not changed. Compared with most real-time quantitative PCR (qPCR) detection methods currently on the market, there is no substantial improvement in sensitivity.

Method used

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  • CRISPR detection primer group for mycoplasma pneumoniae and application of CRISPR detection primer group
  • CRISPR detection primer group for mycoplasma pneumoniae and application of CRISPR detection primer group
  • CRISPR detection primer group for mycoplasma pneumoniae and application of CRISPR detection primer group

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Design of primer sequences for CRISPR detection of Mycoplasma pneumoniae.

[0040] 1. Target sequence selection.

[0041] On the basis of the previous research, the inventors selected the P1 gene sequence region in the Mycoplasma pneumoniae genome that is specific and has no high-frequency SNP sites as the detection target sequence of Mycoplasma pneumoniae after multiple screenings and comparisons. The sequence of SEQ ID NO.1 is as follows:

[0042] 5’-TGATCTCGCCAACGCTCCCATTAAGCGGAGCGAGGAGTCGGGTCAGTCCGTCCAACTCAAGGCGGACGATTTTGGTACTGCCCTTTCCAGTTCGGGATCAGGCGGCAACTCCAATCCCGGTTCCCCCACCCCCTGAAGGCCGTGGCTTGCGACTGAGCAAATTCACAAGGACCTCCCCAAATGATCCGCCTCGATCCTGATTCTGTACGATGCGCCTTATGCGCGCAACCGTACCGCCATTGACCGCGTTGATCACTTGGATCCCAAGGCCATGACCGCGAACTATCCGCCCAGTTGAAGAACGCCCAAGTGAAACCACCACGGTTTGTGGGACTG-3’(SEQ ID NO.1)。

[0043] The standard sample to be tested in the following examples is the plasmid (synthesized by Aiji Biotechnology Co., Ltd.) inserted with the above-mentioned Mycoplasm...

Embodiment 2

[0050] 1. The amplification efficiency screening of RPA amplification primers.

[0051] In order to screen the RPA amplification primer of Cas13a, insert the plasmid of the above-mentioned Mycoplasma pneumoniae P1 gene sequence in the above-mentioned embodiment 1 as the standard sample to be tested, extract the DNA group of the standard sample to be tested (i.e. the Mycoplasma pneumoniae genomic DNA) according to conventional methods, The screening primers include the above-mentioned primer pair 1, primer pair 2, primer pair 3, primer pair 4, and primer pair 5, wherein the template concentration is 1000 copies / μl.

[0052] 1.1 Method.

[0053] Amplify the target sequence by RPA, and use DEPC-treated water without impurity RNA, DNA and protein as a negative reference, select conventional reagents, and proceed as follows:

[0054] 1) RPA amplification

[0055] RPA reaction system is 50 μL: including RPA upstream primer 0.5-2.5 μL (concentration 10 μM), RPA downstream primer 0....

Embodiment 3

[0080] In this example, sensitivity detection is performed based on RPA amplification, T7 in vitro transcription and Cas13a.

[0081] The plasmid DNA inserted with the above-mentioned Mycoplasma pneumoniae P1 gene sequence was used as a template, and the calculated dilutions were 3000copies / μL, 300copies / μL, 30copies / μL, 3copies / μL, and 0copy / μL, a total of 5 gradients were used as templates for sensitivity detection.

[0082] 1. Method.

[0083] Referring to the method in Example 2 above, after RPA amplification, T7 transcription and CRISPR reaction were performed.

[0084] 2. Results.

[0085] ABI7500 fluorescence detector was used to detect the fluorescence of the amplified products. Among them, the result as image 3 As shown, it means that the detection limit of crRNA-1 is 30copies.

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Abstract

The invention relates to a CRISPR (clustered regularly interspaced short palindromic repeat) detection primer group for mycoplasma pneumoniae and an application of the CRISPR detection primer group, and belongs to the technical field of gene detection of a CRISPR technology. The primer group comprises an amplification primer pair and crRNA (CRISPR-derived ribonucleic acid), wherein the amplification primer pair is used for amplifying a sequence shown as SEQ ID NO. (sequence identifier number) 1 of the mycoplasma pneumoniae; crRNA comprises an anchor sequence and a guide sequence; the anchor sequence specifically identifies a Cas protein; and the guide sequence is matched with a target sequence segment in the sequence of SEQ ID NO. 1. The primer group detects the mycoplasma pneumoniae through the CRISPR technology; the detection time of the mycoplasma pneumoniae is shortened; and the detection can be accomplished within 60min. A specific sequence combination obtained by screening acts as the primer group for detection, the primer group has the advantages of high sensitivity and strong specificity, and a limit of detection of the primer group can reach 30 copies. The primer group isused for the CRISPR detection of the mycoplasma pneumoniae, is independent of a complicated variable temperature amplification instrument such as a qPCR (quantitative polymerase chain reaction) instrument, and a CRISPR-Cas technology has wide application prospects in instant diagnosis of the mycoplasma pneumoniae.

Description

technical field [0001] The invention relates to the technical field of gene detection based on CRISPR technology, in particular to a CRISPR detection primer set for Mycoplasma pneumoniae and its application. Background technique [0002] Mycoplasma pneumoniae infection (Mycoplasma pneumonia infection) exists widely in all parts of the world. The infection is transmitted from person to person through the droplets sprayed by patients when they cough. About 80% of patients will have obvious clinical manifestations, usually manifested as pharyngitis, tracheobronchitis, reaction Acute airway disease / wheeze or nonspecific upper respiratory syndrome. The incubation period of Mycoplasma pneumoniae is 2 to 4 weeks, so infection in certain populations can last for several weeks. Mycoplasma pneumoniae disease usually occurs in densely populated places such as military bases, boarding schools, and summer camps. The family infection rate of children is 84%, while that of adults is 41%. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/35
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2521/301C12Q2563/107
Inventor 许腾曾伟奇杨敏玲徐学中陈文景李永军王小锐苏杭
Owner 广州微远医疗器械有限公司
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