Method for detecting pathogens infected by cerebrospinal fluid based on PCR and nanopore sequencing

A pathogen and sequencing data technology, applied in the field of bioinformatics, can solve problems such as inability to culture viruses, inability to fully meet clinical needs, long cycle, etc., and achieve high clinical application value

Active Publication Date: 2021-08-03
PEOPLES HOSPITAL PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Meningitis pathogens include bacteria, fungi, viruses, etc. Traditional culture methods have a long period of time and low sensitivity, and conventional clinical microbiology laboratories cannot culture viruses, which cannot fully meet clinical needs

Method used

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  • Method for detecting pathogens infected by cerebrospinal fluid based on PCR and nanopore sequencing
  • Method for detecting pathogens infected by cerebrospinal fluid based on PCR and nanopore sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Establishment of a method for high-throughput simultaneous detection of which pathogens are infected in multiple cerebrospinal fluid samples

[0036] 1. Obtaining RNA from cerebrospinal fluid

[0037] 1. Collect cerebrospinal fluid (avoid contamination during collection), and store in DNA / RNA Shield (Zymo, Catalog Code R1100-50).

[0038] 2. After completing step 1, cut the cerebrospinal fluid into small pieces to obtain the target sample.

[0039] 3. Take the Glass PowerBead Tube, add 200 μL of the target sample obtained in step 2 and 600 μL of PM1 / β-ME, and place it in a Qiagen homogenizer for 10 minutes at maximum speed; centrifuge at 13,000 g for 1 minute at room temperature, and collect the supernatant.

[0040] 4. Take a 2ml collection tube, add the supernatant collected in step 3 and 150 μL IRS liquid, vortex and mix, incubate at 4°C for 5 minutes; then centrifuge at 13000g for 1 minute, and collect the supernatant (avoid contact with the precipitate)...

Embodiment 2

[0112] Embodiment 2, the method accuracy that detection embodiment 1 establishes

[0113] Sample 1 to be tested is the cerebrospinal fluid of patient 1 who has been clinically confirmed to be infected with Neisseria meningitidis.

[0114] Sample 2 to be tested is the cerebrospinal fluid of patient 2 who has been clinically confirmed to be infected with Cytomegalovirus.

[0115] Sample 3 to be tested is the cerebrospinal fluid of patient 3 who has been clinically confirmed to be infected with Listeria monocytogenes.

[0116] The sample 4 to be tested is the cerebrospinal fluid of the healthy person 1 .

[0117] The sample 5 to be tested is the cerebrospinal fluid of the healthy person 2 .

[0118] Test sample 1-test sample 5 according to the method established in Example 1. The results showed that test sample 1 was infected with Neisseria meningitidis, test sample 2 was infected with Cytomegalovirus, test sample 3 was infected with Listeriamonocytogenes, test sample 4 and test...

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Abstract

The invention discloses a method for detecting pathogens infected by cerebrospinal fluid based on PCR (polymerase chain reaction) and nanopore sequencing. Experiments prove that the method provided by the invention is used for detecting the cerebrospinal fluid, and the method can be used for accurately judging which one or more of pathogens for infecting the central nervous system is or are selected from the group consisting of Enterovirus, Lymphotic virus, Neisseria meningiosis, Cytomegalovirus, Mycobacterium tubers, Nocardia Farcina, Herpes simplex virus 1, Human herpes virus 6, Listeria monocytogenes and Herpes simplex virus 2. The invention provides accurate and rapid etiological diagnosis for central nervous system infection, and has very high clinical application value.

Description

technical field [0001] The invention belongs to the field of bioinformatics, and in particular relates to a method for detecting pathogens infected with cerebrospinal fluid based on PCR and nanopore sequencing. Background technique [0002] Meningitis is a serious, life-threatening inflammation of the meninges and subarachnoid spaces that, due to their anatomical proximity, may also involve the cerebral cortex and spinal cord and require immediate medical attention and management. Meningeal inflammation causes vasospasm and may lead to thrombosis of cerebral arterioles and arteries and possible cerebral venous occlusion. Various inflammatory products of bacteria and neutrophils can cross the blood-brain barrier, causing neuronal necrosis or compression of cranial nerves. Meningitis occurs worldwide, develops in individuals of all ages, and causes high morbidity and mortality. [0003] Meningitis pathogens include bacteria, fungi, viruses, etc. Traditional culture methods h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6869C12N15/11
CPCC12Q1/705C12Q1/689C12Q1/6869C12Q2600/16C12Q2531/113C12Q2537/143C12Q2535/122C12Q2565/631C12Q2537/165Y02A50/30
Inventor 陈宏斌王辉
Owner PEOPLES HOSPITAL PEKING UNIV
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