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Drug-resistant bacterium detection method based on nucleic acid molecule machine colorimetry

A detection method and technology of nucleic acid molecules, applied in the detection field, can solve problems such as time-consuming, cumbersome detection process, and inability to quantitatively detect, and achieve the effects of high binding efficiency, large false positive signals, and short time

Pending Publication Date: 2020-07-31
苏州朴衡科技有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Bacterial culture method is the absorbency method of clinical bacteria detection, but the detection process is cumbersome and time-consuming. The culture method takes up to 24-72 hours (it is helpless for bacteria that are difficult to culture or grow slowly), and it cannot reveal the mechanism of drug resistance. , not suitable for rapid detection of bacteria
PCR (polymerase chain reaction) and genome sequencing have high sensitivity and good specificity, and can achieve trace-level detection, which is of great significance for the detection of low-level bacteria. The technology relies on known reference sequences of drug-resistant genes, so it cannot analyze unknown Drug resistance, and usually cannot be detected quantitatively, so generally only as an adjunct
These conventional bacterial detection methods have certain limitations in terms of sensitivity, specificity, detection speed, cost, and high-throughput detection and analysis of bacteria. Therefore, it is urgent to establish a simple, fast, sensitive, and visual bacterial detection method with important meaning

Method used

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  • Drug-resistant bacterium detection method based on nucleic acid molecule machine colorimetry
  • Drug-resistant bacterium detection method based on nucleic acid molecule machine colorimetry
  • Drug-resistant bacterium detection method based on nucleic acid molecule machine colorimetry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] The preparation of embodiment 1 double probe

[0123] The traditional method uses sulfhydryl-modified DNA to connect to the surface of the gold ball and uses the salt aging method, which often takes one to two days, which is time-consuming and laborious. The present invention adopts a new double-probe preparation method: a certain concentration of citrate is directly added to the double-probe that responds specifically to bacteria, and the pH in the solution is controlled to be 3, thereby weakening the static electricity between AuNPs and SH-DNA Repulsion, enhance the binding probability of SH-DNA and AuNPs.

[0124] In this example, 4 μl of sulfhydryl-modified probe chains was mixed with 40 μl of AuNPs (60 nM) at a volume ratio of 1:1, 4.88 μl of citrate (100 mM, pH=3) was added and vortexed rapidly for 2 min. And make the final concentration of citrate 10mM. After standing in the dark for 15 minutes, centrifuge at 10,000 r for 10 minutes, discard the supernatant, an...

Embodiment 2

[0127] Embodiment 2 specificity experiment

[0128] MRSA was used as the experimental group, and PBS, Escherichia coli (E.coli), Shigella funeri (Shigella) and Pseudomonas aeruginosa (Paeru) were used as the control group. Take 5 reaction mixtures, each reaction mixture includes: 100μl dual probe (30nM), 30μl double-stranded core (RNA1-RNA2-Aptamer), 2μl EXOIII and 20μl 10×buffer, and then add to the 5 reaction mixtures respectively 50 μl of different types of the above bacterial suspension (MRSA, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa) and 50 μl of PBS were prepared as samples. The bacterial concentration in all samples was controlled at 10 8 CFU / ml (except PBS group). After measuring the initial absorbance of the reaction mixture solution with an ultraviolet spectrophotometer, the reaction mixture solution was incubated in a 37° C. incubator for 2 h, and the absorbance of the reaction mixture was measured with an ultraviolet spectrophotometer.

[0129...

Embodiment 3

[0131] The detection of embodiment 3 MRSA

[0132] The high-concentration MRSA cultured in liquid medium was centrifuged and washed three times, redispersed with PBS, and bacterial suspensions of various dilutions were obtained after gradient dilution. Take nine copies of 100 μl double-probe (30nM), add 50 μl bacterial suspension with different concentration gradients, 30 μl double-stranded nucleic acid (RNA1-RNA2-Aptamer), 2 μl EXOIII, and 20 μl 10× buffer to the control reaction system The final concentration of EXOIII was 15U / L and the final concentrations of the serially diluted bacterial solution were 0, 1, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , and 10 8 CFU / ml to obtain a reaction mixture solution.

[0133] After measuring the initial absorbance of the reaction mixture solution with an ultraviolet spectrophotometer, the above reaction mixture solution was incubated in a 37° C. incubator for 2 hours, and the absorbance of the reaction mixture was measured wi...

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Abstract

The invention discloses a drug-resistant bacterium detection method based on nucleic acid molecular machine colorimetry. The method comprises the following steps: (1) constructing double probes with bacterial specific response: mixing sulfydryl-modified double probe chains 1 and 2 with a nano material, adjusting the pH value, and then performing standing and centrifuging to obtain double Probe1 and Probe2 with bacterial specific response; (2) preparing a reaction solution: storing the double probes with the bacterial specific response and a reaction mixture into a centrifugal tube to prepare the reaction solution, wherein the reaction mixture comprises a double-stranded nucleic acid (RNA1-RNA2-Aptamer) and EXOIII; and (3) detecting bacteria: mixing a bacteria solution to be detected with the reaction solution prepared in the step (2) according to a certain volume ratio, carrying out incubation reaction, measuring ultraviolet absorptivity, and determining bacteria types. According to the method, signal cyclic amplification is carried out at a double enzyme digestion rate, common drug-resistant bacteria in hospitals can be identified more quickly, the whole detection process is shorter than 4 hours, and sensitivity and accuracy are achieved; and the method has the advantages of being good in specificity, simple to operate and visible to the naked eyes, and lays a foundation for research and development of rapid bacterial diagnosis kits.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a drug-resistant bacteria detection method based on a nucleic acid molecular machine colorimetric method. Background technique [0002] Antibiotic resistance is an imminent threat to global health. Since Alexander Fleming, a British bacteriologist, discovered penicillin in 1928 and penicillin G was first used clinically in 1940, antibiotics have been used for 77 years. Over the past 20 years, antibiotics have made great contributions to human health and medical progress. However, the emergence of drug-resistant bacteria may bring mankind back to the "pre-antibiotics" dark age when no drugs are available for many infections. Based on the rise of drug resistance in 6 pathogenic bacteria, it is estimated that unless action is taken, the number of deaths caused by AMR can increase to 10 million people per year, and the cumulative cost to the global economy will reach 100 trillion U...

Claims

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Application Information

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IPC IPC(8): C12Q1/6816C12Q1/14C12Q1/06C12R1/445
CPCC12Q1/6816Y02A50/30
Inventor 裴昊邹奎李丽
Owner 苏州朴衡科技有限公司
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