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Human mycoplasma detection system based on RPA-CRISPR/Cas12a and application thereof

A technology of mycoplasma hominis and detection system, which is applied in the field of microbial detection, can solve the problems that hinder the large-scale promotion of detection methods, difficulty in separation and culture, time-consuming and labor-intensive, etc., and achieve visual detection with naked eyes, high specificity and sensitivity, and shorten time. Effect

Pending Publication Date: 2022-04-26
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other pathogens related to the reproductive tract, Mycoplasma hominis has no cell wall, grows slowly, and has strict requirements for culture and identification, making isolation and culture more difficult and time-consuming.
qPCR is a high-sensitivity and high-specificity nucleic acid detection technology, but this method requires professionals to operate and requires complex instruments and equipment. Not all laboratories have such detection conditions, and it is time-consuming and costly. hinder the wide-scale promotion of this detection method

Method used

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  • Human mycoplasma detection system based on RPA-CRISPR/Cas12a and application thereof
  • Human mycoplasma detection system based on RPA-CRISPR/Cas12a and application thereof
  • Human mycoplasma detection system based on RPA-CRISPR/Cas12a and application thereof

Examples

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Effect test

Embodiment 1

[0062] 1. Construction and implementation of a nucleic acid detection method for Mycoplasma hominis based on RPA amplification and CRISPR-Cas12a

[0063] 1.1 Mycoplasma hominis gene sequence analysis to find the target of Mycoplasma hominis CRISPR / Cas nucleic acid detection

[0064] Methods: According to literature reports, the specific conserved gene sequences of Mycoplasma hominis were found, namely 16srRNA and gap gene, and the corresponding constant temperature amplification primers were designed for verification.

[0065] 1.2 Design and synthesis of constant temperature amplification primers

[0066] The 16sRNA and gap gene sequences of Mycoplasma hominis were found in the NCBI database, and Primer 5 was used to design specific primers, and at the same time, the designed primers were specifically screened on NCBI. The sequence of the primers was required: avoid unusual sequences in the primers, Such as a long sequence consisting entirely of a specific nucleotide, or a sh...

Embodiment 2

[0169] Establishment of a method for detecting Mycoplasma hominis based on RPA-CRISPR / Cas12a-colloidal gold lateral flow chromatography and its application method ( Figure 12 ②):

[0170] 2.1 Construction of colloidal gold lateral flow chromatography test strip:

[0171] For details, see Figure 7 , the present invention provides a method of combining RPA / Cas12a with immunocolloidal gold test strips to detect Mycoplasma hominis. The nucleic acid colloidal gold test strips include a PVC backing film, a sample pad, a binding pad, a nitrocellulose film and an absorbent pad . The nitrocellulose membrane at the detection line on the test strip, the nitrocellulose membrane on the quality control line is coated with streptavidin, and the goat anti-rabbit secondary antibody (FITC antibody combined with fluorescein isothiocyanate) is prepared. The secondary antibody is combined with the primary antibody of fluorescein isothiocyanate FITC and simultaneously immobilized colloidal gol...

Embodiment 3

[0194] Construction of a method for detecting Mycoplasma hominis based on RPA-CRISPR / Cas12a-time-resolved europium-labeled microsphere immunochromatographic test strips and its application method ( Figure 12 ③).

[0195] 3.1 Construction of time-resolved europium-labeled microsphere immunochromatographic test strips

[0196] The present invention is a method for combining RPA / Cas12a to detect Mycoplasma hominis. The sample to be tested containing the RPA-Cas12a reaction system is dropped on the sample pad, and capillary action chromatography is used. When there is no pathogenic nucleic acid to be detected, the probe is not detected. After being cleaved, the complex formed by the carboxylated europium chelate nano fluorescent microspheres coupled anti-FITC antibody and FITC-Biotin probe is captured by streptavidin on the quality control line (C line) and fixed on the C line superior( Figure 10 ). When the ssDNA nucleic acid sequence of the pathogen to be detected exists, t...

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Abstract

The invention discloses a mycoplasma hominis detection system based on RPA-CRISPR / Cas12a and application of the mycoplasma hominis detection system, and relates to the field of microbiological detection. The detection system comprises an RPA (recombinase polymerase amplification) primer pair, a probe, crRNA (complementary Ribonucleic Acid) and AsCas12a protein, the sequences of the RPA amplification primer pair are as shown in SEQ ID NO: 3 and SEQ ID NO: 4; the sequence of the probe is as shown in SEQ ID NO: 16; the crRNA is a sequence as shown in any one of SEQ ID NO: 10 to SEQ ID NO: 12. According to the three rapid nucleic acid detection methods for the mycoplasma hominis, which are constructed on the basis of the RPA-CRISPR / Cas12a detection system, the detection sensitivity is increased, and the method disclosed by the invention shows extremely high specificity, so that a new technical platform is provided for the detection of the mycoplasma hominis.

Description

technical field [0001] The invention relates to the field of microbial detection, in particular to a detection system for Mycoplasma hominis based on RPA-CRISPR / Cas12a and its application. Background technique [0002] Mycoplasma is the smallest self-replicating prokaryote. More infections include Ureaplasma urealyticum and Mycoplasma hominis. Mycoplasma hominis (Mycoplasma homini, M.homini) is one of the main pathogens of genitourinary tract infection. It mainly produces ammonia by decomposing arginine, which causes immune stimulation and human harm to host cells, causing urogenital tract and external genital tract infection. . Especially in female patients, it can cause female vaginitis, birth canal infection and other diseases. Scholars at home and abroad have found that with the openness of human sexual consciousness, non-standard use of antibiotics, and relatively backward clinical detection technology, some patients are difficult to diagnose and affect the effect of...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2521/327C12Q2563/107C12Q2565/625C12Q2521/507C12Q2522/101Y02A50/30
Inventor 郝文波黄颖而陈佳灵
Owner SOUTHERN MEDICAL UNIVERSITY
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