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Rapid pathogenic microorganism detection method and kit thereof

A technology of pathogenic microorganisms and detection methods, applied in the field of rapid detection methods and kits for pathogenic microorganisms, can solve the problems of difficult interpretation of sequencing results, long sequencing time, and short sequencing read length, etc., and achieve the effect of improving the success rate of library construction

Pending Publication Date: 2022-03-18
WUHAN EASYDIAGNOSIS BIOMEDICINE
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0006] For the detection of pathogenic microorganisms, the current general method is to use the second-generation sequencing platform for metagenomic sequencing. This platform has long sequencing time, short sequencing read length, large amount of human genome DNA contamination in the sequencing results, high sequencing data volume and poor sequencing results. more difficult questions

Method used

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  • Rapid pathogenic microorganism detection method and kit thereof
  • Rapid pathogenic microorganism detection method and kit thereof
  • Rapid pathogenic microorganism detection method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 Comparison test between the present invention and Nanopore official process

[0066] For the database construction technology, Nanopore official also provided part of the experimental process. In order to compare the difference between the experimental results of the detection method of the present invention and the official Nanopore process, a comparative test was carried out using standard products. The main comparative test conditions are as follows:

[0067] Table 6 Comparison of test conditions between the method of the present invention and the Nanopore official process

[0068] Experimental items official process this invention Nucleic acid amount 30ng 50pg~100ng Amplification process One Tube Direct Expansion two-stage amplification connection method ligase-independent ligation ligase ligation

[0069] When using the DNA extracted from a single cultured microorganism, according to the required 30ng test, ...

Embodiment 2

[0074] Embodiment 2 The inventive method standard substance detection limit test

[0075] For clinical applications, the detection limit is a key indicator for judging the experimental effect. The currently known methods require the number of microorganisms to be extracted to reach 20 or more copies in order to be detected normally. For standard microbial strains, the following 20 , 10, and 4 copy number detection tests, the specific detection results are as follows:

[0076] Table 8 standard detection limit experimental results

[0077]

[0078]

[0079] Microbiological standards are microbial solutions diluted to 100 copies / mL, including Candida albicans, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Enterococcus faecalis, Acinetobacter baumannii, Candida glabrata, EBV virus and CMV For viruses, 200uL, 100uL, and 40uL of standard bacterial solutions were extracted respectively during nucleic acid extraction. After extraction, pathogenic microorg...

Embodiment 3

[0080] Embodiment 3 clinical blood sample detection

[0081] Take 10 cases of whole blood samples with clear pathogenic microorganisms in clinical blood culture, take 200uL whole blood to extract nucleic acid, and then perform library construction and sequencing according to the method of the present invention. All 10 cases of samples were normally built and sequence data were obtained. The specific detection results are as follows :

[0082] Table 9 The detection results of different clinical blood samples

[0083] sample blood culture results Is it consistent blood 1 Escherichia coli unanimous blood 2 Staphylococcus epidermidis not detected blood 3 Pneumonia Kleb unanimous blood 4 Staphylococcus haemolyticus unanimous blood 5 Staphylococcus aureus unanimous blood 6 Enterococcus faecium unanimous blood 7 Pseudomonas aeruginosa unanimous blood 8 Streptococcus pneumoniae unanimous blood 9...

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Abstract

The invention discloses a rapid pathogenic microorganism detection method adaptive to a Nanopore sequencing platform and a kit thereof. Detection of pathogenic microorganisms can be completed within 4-6 hours. According to the invention, a specific primer is designed for a detection target, and the enrichment of nucleic acid is realized through two rounds of PCR amplification; a universal sequence is added to the 5'end of a specific primer, a first-round PCR product does not need to be purified and is directly used for second-round amplification, phosphorylation is added to the tail end of a primer with a universal tag sequence during second-round amplification, the generated amplification product can be directly subjected to TA connection of a sequencing joint, the library building step is greatly simplified, and the experiment time is shortened. According to the method, the problem of human genome DNA pollution in pathogenic microorganism detection is solved, the whole fragment can be directly detected without splicing amplified long fragment data by using a Nanopore sequencing platform with a long read length, and the average length of data sequenced by the method is 1.6 k and is increased by more than 10 times compared with that of a second-generation platform; the accuracy and the stability of pathogenic microorganism judgment can be greatly improved.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a rapid detection method for pathogenic microorganisms and a kit thereof. Background technique [0002] Microorganisms that are pathogenic to humans and animals are called pathogenic microorganisms, also known as pathogens, including viruses, bacteria, rickettsia, mycoplasma, chlamydia, spirochetes, fungi, and actinomycetes. These pathogenic microorganisms can cause infections, allergies, tumors, dementia and other diseases, and are also one of the main factors that endanger food safety. Diseases such as SARS, highly pathogenic avian influenza, and West Nile virus infection that have emerged in recent years are highly contagious and often cause worldwide pandemics. Therefore, the detection of pathogens must be fast and accurate. Conventional etiological detection methods are cumbersome to operate, the detection cycle is long, and the requirements for the technical l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686C12Q1/6869C12Q1/689C12Q1/6895C12Q1/70C12N15/11
CPCC12Q1/686C12Q1/6869C12Q1/689C12Q1/701C12Q1/6895C12Q2600/16C12Q2537/143C12Q2525/113C12Q2535/122
Inventor 方涛袁凯王颖龙志成
Owner WUHAN EASYDIAGNOSIS BIOMEDICINE
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