Kit for synchronously detecting twenty-three meningitis pathogens and detection method of kit

A technology for synchronous detection and meningitis, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/testing, etc., can solve the problems of low diagnostic efficiency, uncertain sensitivity, high technical cost, etc., and achieve saving production Cost and detection cost, good sensitivity and specificity, avoid the effect of low specificity

CN103074448BActive Publication Date: 2014-03-26NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2014-03-26

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Abstract

The invention discloses a kit for synchronously detecting twenty-three meningitis pathogens and a detection method of the kit. The kit comprises DEPC (diethylpyrocarbonate) water, a 5*RT (reverse transcription) buffer, a reverse transcription primer, a reverse transcriptase, an X solution, a 10*PCR (polymerase chain reaction) buffer, a PCR primer, a 25mM magnesium chloride solution, a DNA (deoxyribonucleic acid) polymerase and a positive control, and is characterized in that the reverse transcription primer comprises RT amplification primers of the twelve meningitis pathogens and a human RNA (ribonucleic acid) internal reference, and has a gene sequence shown as SEQ ID NO. 1-13 (sequence identifier number 1-13), and the PCR primer comprises forward and reverse PCR amplification primers of the rest eleven meningitis pathogens, a human DNA internal reference and a reaction internal reference, and PCR amplification primers of the twelve meningitis pathogens and the human RNA internal reference, and has a gene sequence shown as SEQ ID NO. 14-52. The kit and the detection method have the advantages of high specificity, sensitivity, flux and reliability, low cost, and no false negative results.
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Description

technical field

[0001] The present invention relates to a detection kit for pathogens of encephalitis and meningitis and a detection method thereof, in particular to a kit for synchronously detecting twenty-three pathogens of encephalitis and meningitis based on a GeXP multiple gene expression genetic analysis system and detection thereof method. Background technique

[0002] Brain fever (meningitis) is an infection of the delicate meninges, or meninges (the membrane between the skull and the brain). The disease is often accompanied by complications from bacterial or viral infection of any part of the body, such as ear, sinus, or upper respiratory infections. The pathogens causing encephalitis and meningitis are diverse, and the scope and degree of lesions vary greatly, and the clinical manifestations are very complicated. The current clinical diagnostic methods such as EEG, imaging, pathogen culture, serological examination and other methods cannot quickly and accurately ...

Examples

specific Embodiment 1

[0040] The present invention is a kit for synchronous detection of 23 types of encephalitis and meningitis pathogens, comprising:

[0041] 1) RT primer (reverse transcription primer RT primer Mix)

[0042] 2) PCR Primer (PCR Primer Mix)

[0043] 3) 25mM magnesium chloride (25mM MgCl2)

[0044] 4) Reverse transcriptase

[0045] 5) DNA polymerase (Taq DNA Polymerase)

[0046] 6) Solution X (Solution X)

[0047] 7) 10x PCR buffer (10x PCR Buffer)

[0048] 8) 5×RT buffer (5x RT buffer)

[0049] 9) Positive Control (Positive Control)

[0050] 11) RNase / DNase-free ultrapure water (DNase / Rnase Free ddH2O)

[0051] 12) Positive control substances (DNA fragments with specific sequences, used for quality control of the entire reaction system)

[0052] The above-mentioned reverse transcription primers include the RT amplification primers of twelve kinds of encephalitis and meningitis pathogens in the following table and the RT amplification primers of human RNA internal reference...

specific Embodiment 2

[0062] The invention is a detection method for synchronously detecting 23 kinds of encephalitis and meningitis pathogens, the detected encephalitis and meningitis pathogens include Japanese encephalitis virus, Powassen virus, West Nile virus, herpes simplex virus type 1, simplex Herpesvirus type 2, varicella-zoster virus, African lymphoma virus, human cytomegalovirus, all enteroviruses, coxsackievirus A16, enterovirus 71, mumps virus, Nipah virus, meninges Neisseria, Haemophilus influenzae type b, Staphylococcus, Streptococcus pneumoniae, Streptococcus suis, Escherichia coli, Cryptococcus, Toxoplasma gondii, cysticercosis, Plasmodium, Mycoplasma pneumoniae, etc. (see Table 1), collect patient samples (cerebrospinal fluid, blood, etc.) extract nucleic acid, use patient nucleic acid as a template for reverse transcription and PCR reactions, and finally separate samples by capillary electrophoresis. The specific steps are as follows:

[0063] 1. Production of a kit for the simult...

specific Embodiment 3

[0091] Detection Kit Sensitivity and Specificity Analysis

[0092] Sensitivity analysis: After diluting the positive control according to a certain copy number ratio, it is detected by PCR amplification and capillary electrophoresis until no signal is detected. The copy number is the lowest detection line, which is the sensitivity of the kit. The highest sensitivity can detect 40 copies.

[0093] Specificity analysis: Single-plex PCR amplification is detected as a single peak of the target fragment size by capillary electrophoresis.