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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

Active Publication Date: 2014-03-26
NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also the following disadvantages: 1) Long time-consuming: generally 3-5 days or longer; 2) Biopsy of brain tissue from patients with encephalitis is difficult to be accepted by patients and their families; 3) Low diagnostic efficiency: some viruses cannot or are extremely difficult to diagnose Difficult to cultivate, different viruses require different isolation methods, it is difficult for general hospitals to carry out, it cannot achieve the purpose of diagnosis, and it seriously lags behind clinical treatment
However, there are the following disadvantages: 1) False positives are prone to occur: due to the operation of washing and antigen coating, or cross-reaction due to similar antigenic surface determinants, false positives are prone to occur; 2) Time-consuming and labor-intensive: making antibodies is very expensive Time-consuming and labor-intensive work; 3) Uncertain sensitivity: the sensitivity of the experiment depends on the quality of the antibody, and the quality of each antibody is different, and the quality is difficult to control; 4) Unable to detect mutated viruses
But there are also the following disadvantages: 1) Low throughput: only one pathogenic component can be detected at a time. If multiple pathogens need to be detected, multiple PCR instruments need to work at the same time. The efficiency is low and the cycle is long. 2) The cost is relatively high: because only one pathogen can be detected at a time, when a sample needs to detect multiple pathogens at the same time, it must be tested one by one, and the cost is relatively high
Advantages: 1) High-throughput parallel detection: When a sample needs to detect multiple pathogenic bacteria at the same time, all the results can be obtained in one experiment; 2) Simple and fast operation: the whole detection can basically produce results in 4-8 hours; but There are also the following disadvantages: 1) The technology is expensive and complicated: each sample requires a chip, and the cost is more than ¥1000 / sample, which is not conducive to large-scale promotion; 2) The synthesis and immobilization of probes are more complicated, especially the production of high-density Probe array is the main rate-limiting step; 3) It cannot be accurately quantified and the repeatability is poor; 4) The sensitivity is low: the chip method requires a large amount of nucleic acid, and generally requires multiple PCR amplification first. Dimers, hairpin structures, or different Tm values ​​lead to different amplified target fragment efficiencies, which in turn affect detection sensitivity; 5) Due to the variety of chips, it is difficult to formulate a unified quality control standard
[0018] At present, there are no relevant research reports on the kits and detection methods for the simultaneous detection of 23 encephalitis and meningitis pathogens based on the GeXP multiple gene expression genetic analysis system at home and abroad

Method used

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

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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.

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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.

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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/14C12Q1/04C12R1/93C12R1/36C12R1/21C12R1/44C12R1/46C12R1/19C12R1/35C12R1/90C12R1/01
Inventor 吴勇黄迎彬南丽
Owner NINGBO HEALTH GENE TECHNOLOGIES CO LTD
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