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

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 ...

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

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