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A multiplex PCR kit for detecting pathogens of bacterial meningitis

A meningitis and bacterial technology, applied in the field of multiplex PCR kits, can solve the problems of reducing multiplex PCR detection throughput, limitations, etc., and achieve the effects of reducing non-specific products, reducing detection costs, and saving reagent usage

Active Publication Date: 2019-05-17
上海捷诺生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As the number of primer pairs increases, the inhibitory effect between primers reduces the detection throughput of multiplex PCR. In the actual application of multiplex PCR technology, since multiple primers are added in the same reaction tube, how to improve the specificity of Non-specific amplification, how to reduce dimers formed between multiple primers, how to regulate the concentration of PCR reagents and reaction conditions, so that each target fragment can be effectively amplified, etc., are all limited. application of multiplex PCR

Method used

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  • A multiplex PCR kit for detecting pathogens of bacterial meningitis
  • A multiplex PCR kit for detecting pathogens of bacterial meningitis
  • A multiplex PCR kit for detecting pathogens of bacterial meningitis

Examples

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Embodiment 1

[0027] Example 1 Specificity and Sensitivity Detection of Multiplex System

[0028] 1. Specific detection of multiplex system

[0029] A multiplex PCR kit for detecting bacterial meningitis pathogens, including conventional multiplex PCR components, and chimeric primers and public primers for common meningitis-infected bacteria; the sequences of chimeric primers and public primers are shown in Table 1, and conventional multiplex PCR components include Taq polymerase, dNTPs, MgCl 2 , PCR buffer, deionized water; positive control primer pair, which can be used to detect the quality of the DNA template and the amplification efficiency of the reaction system, the specific sequence is: SEQ ID NO.14, 5'-AGCGGGAAATCGTGCGTGA-3'; SEQ ID NO.15 , 5'-AGTGAGGACCCTGGATGTGA-3'.

[0030] Table 1 Chimeric primers and public primers for bacterial pathogens of meningitis infection

[0031]

[0032] Specific detection of multiplex systems: use the PCR method of multiple primers + single templ...

Embodiment 2

[0035] Example 2 Detection of clinical cerebrospinal fluid specimens

[0036] Sixty cerebrospinal fluid (CSF) samples suspected of central nervous system infection were collected, and the genomes of the collected CSF samples were extracted according to the extraction method of Gram-positive bacteria in the DNAmini Kit (Catalog No. 51304) of QIAgen. The multiplex PCR kit for detecting bacterial meningitis pathogens of the present invention is used to detect the genomic DNA of the extracted specimen. The 25 μL reaction system used in the multiplex PCR includes: 5 μL of 10 μM common primers, 0.05 μM (each) chimeric primer mixture 5 μL, 5 U / μL Taq polymerase 0.15 μL, 25 mM MgCl 2 2μL, 10mM dNTP 0.5μL, 10×TaqBuffer 2.5μL, DNA template 1μL, sterilized ddH 2 O 8.85 μL; PCR reaction conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 63.5°C annealing for 30 s, 72°C extension for 45 s, a total of 10 cycles; 72°C extension for 3 min; 95°C denaturation for 30 s, 55...

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Abstract

The invention discloses a multiplex PCR kit for detecting bacterial meningitis pathogens. Six types of bacterial meningitis pathogens, namely streptococcus pneumoniae (SP), streptococcus agalactiae (GBS), haemophilus influenzae (HI), listeria monocytogenes (LM), staphylococcus aureus (SA) and neisseria meningitidis (NM) can be rapidly detected. The invention discloses a 'public primer'-mediated multiplex PCR method. The method concretely comprises the steps of constructing a plasmid template, screening a public primer, designing a multi-primer and carrying out multiplex PCR system detection; a two-step annealing temperature method is combined, so that specificity, sensibility and detection throughput of a multiplex PCR system are improved; the multiplex PCR method has a certain containment capability, detection targets can be continuously added, and the detection throughput of multiplex PCR can be beneficially improved. The multiplex PCR kit disclosed by the invention can be used for effectively detecting six types of common bacteria, has the advantages of high sensitivity, economy, high speed and simple operation and is beneficial to application and clinical diagnosis.

Description

technical field [0001] The invention belongs to the field of gene detection and specifically designs a multiplex PCR kit for detecting six common pathogens of bacterial meningitis. technical background [0002] Multiplex PCR is a PCR technique that uses multiple pairs of primers to simultaneously amplify multiple target sequences in the same reaction system on the basis of ordinary PCR. Since multiplex PCR was reported, due to its high efficiency, sensitivity, and economic simplicity, it has been widely used in many fields of nucleic acid diagnosis, such as genetic disease diagnosis, mutation and polymorphism analysis, transgene identification, pathogen detection, etc.; multiplex PCR System, generally includes the following reagents: PCR buffer, Taq enzyme, dNTP, MgCl 2 , primers, template and deionized water. [0003] Bacterial meningitis (BM) is an inflammation of the meninges and meninges caused by various bacterial infections. It is a relatively common acute and severe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 孙万平
Owner 上海捷诺生物科技股份有限公司
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