Probes, chip, primers and kit for rapid detection of ophthalmic clinical microorganisms and application thereof

A technology of microorganisms and probes, applied in the field of microorganism detection, can solve the problems of cumbersome process, long return period of results, and inability to provide timely help for daily diagnosis.

Active Publication Date: 2021-04-09
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 16S rDNA sequencing technology relies on large-scale sequencing platforms and equipment, and it cannot be applied on a large scale in hospitals at present, making this technology still rely on the sequencing services provided by third-party companies. sexual help

Method used

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  • Probes, chip, primers and kit for rapid detection of ophthalmic clinical microorganisms and application thereof
  • Probes, chip, primers and kit for rapid detection of ophthalmic clinical microorganisms and application thereof
  • Probes, chip, primers and kit for rapid detection of ophthalmic clinical microorganisms and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Primer Screening

[0083] The strain-specific DNA sequences were screened by genome-wide alignment. Download all the genome sequences of 9 pathogenic bacteria in NCBI, find the homology within the species >95% (most of them can reach more than 97%), and the homology between species <75% (most of them are lower than 70% or basically no homology between species) gene sequence segments. The summary table of genes used for screening primer sequences selected by various strains is shown in Table 3:

[0084] Table 3 Summary table of genes used for screening primer sequences selected by each bacterial species

[0085]

[0086]

Embodiment 2

[0088] Primer Design and PCR Validation

[0089] Firstly, separate primers were designed with primer5 for the DNA segments of each selected strain, and a batch of primers were screened out. Finally, primer selection was performed on the screened primers with DNAstat software to find out a more suitable primer combination. Synthesize the primer, add a fluorescent group (CY3) to the 5' end of the forward primer, so that the PCR product will be fluorescent, and then used for hybridization of the chip of the present invention and then scanned by a scanner to determine whether the hybridization probe is successful .

[0090] The primers screened and verified in Example 2 (as shown in Table 2) have high specificity and can generate readable fluorescent signals after hybridization.

Embodiment 3

[0092] Probe Design and Screening

[0093] The number of probes that can be accommodated by one LCS chip is: 3968 (31*128), in order to carry out probe screening in the largest range, the present invention carries out Tiling array probe design to the PCR product of each bacterium, promptly adopts equal length displacement method, Select a sequence of a certain length (25 bp in the present invention) from the beginning to the end of the target sequence to form a probe combination, wherein the adjacent probe sequences differ by one base until the entire target sequence is covered. According to the NCBI blast results of the PCR product sequence, for the segment with mutations, design multiple probe sequences according to the mutated bases. If a certain probe covers too many mutations (more than 3 mutations in 25bp), abandon this one. probe. Whether the probe is suitable for hybridization needs to be screened by performing a hybridization experiment, and the successfully amplifie...

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Abstract

The invention relates to probes, a chip, primers and a kit for rapid detection of ophthalmic clinical microorganisms and application thereof, and belongs to the technical field of clinical microorganism detection. The probes disclosed by the invention comprise probes for detecting staphylococcus epidermidis, staphylococcus aureus, staphylococcus haemolyticus, pseudomonas aeruginosa, staphylococcus hominis, serratia marcescens, escherichia coli, bacillus subtilis and enterobacter cloacae, respectively. The invention also discloses the primers for amplifying the target strains, and the primers can be used to amplify gene sequence fragments with the intraspecific homology of the target strains > 95% and with the interspecific homology < 75%. The invention further discloses a method for synthesizing hybridization probes on the chip, a method for hybridization reaction and a method for scanning detection. The probes disclosed by the invention are high in specificity, high in positive rate of detecting microorganisms in ophthalmic clinical samples, high in accuracy and short in diagnosis time.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a probe, chip, primer, kit and application for rapid detection of ophthalmic clinical microorganisms. Background technique [0002] Bacterial culture and smear staining were used to identify ophthalmic infection bacteria in the past, but there were problems of long identification time and low positive rate. Eye infection progresses rapidly, and early and timely diagnosis is particularly critical in order to save the patient's vision and reduce damage in time. Relying solely on the morphological judgment of bacteria may also cause misjudgment, and the pathogenic bacteria cannot be qualitatively infected, and only a suspected diagnosis can be made. Most cases of ophthalmic infections are mixed infections with complex bacterial flora, which are usually the result of the joint action of multiple pathogenic bacteria. Traditional culture methods can usually only ident...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6837C12Q1/14C12Q1/10C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6837C12Q2531/113C12Q2563/107C12Q2565/501Y02A50/30C12Q1/6816C12Q1/6844
Inventor 黄钰森张碧凝李文凤刘晴任志超陈华波
Owner SHANDONG EYE INST
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