Kit for synchronously detecting fifteen hemorrhagic fever pathogens and detection method of kit

Abstract

The invention discloses a kit for synchronously detecting fifteen hemorrhagic fever 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 nine hemorrhagic fever pathogens and a human RNA (ribonucleic acid) internal reference, and has a gene sequence shown as SEQ ID NO. 1-10 (sequence identifier number 1-10), and the PCR primer comprises forward and reverse PCR amplification primers of the rest six hemorrhagic fever pathogens, a human DNA internal reference and a reaction internal reference, and PCR amplification primers of the nine hemorrhagic fever pathogens and the human RNA internal reference, and has a gene sequence show as SEQ ID NO. 10-36. 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 invention relates to a detection kit and a detection method for hemorrhagic fever pathogens, in particular to a kit for synchronously detecting fifteen kinds of hemorrhagic fever pathogens based on a GeXP multiple gene expression genetic analysis system and a detection method thereof. Background technique [0002] Fever with hemorrhage is an important infectious disease that endangers human health. It is popular, critical, and has a high fatality rate. It is extremely harmful. Because the disease has fever symptoms, it is often misdiagnosed as upper respiratory tract infection. In addition, routine laboratory tests such as blood biochemical tests, serological tests, and virus isolation cannot quickly and accurately identify various pathogens, resulting in delays in treatment and even the spread of the epidemic. Therefore, there is an urgent need for an efficient diagnostic method for hemorrhagic fever pathogens. [0003] At present, the tradition...

AI Technical Summary

Problems solved by technology

Disadvantages: initial judgment, can not be diagnosed

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 multivariate pathogens

Disadvantages: 1) Long time-consuming: generally 3-5 days or longer; 2) Low diagnostic efficiency: only a single bacterium can be purely cultured; for some pathogens with variable phenotypic characteristics, it is difficult to distinguish with morphological experience; in addition, due to Abuse of antibiotics, the growth of bacteria is inhibited during the detection process, resulting in false negative results; 3) Huge workload: Since every bacteria in each sampling sample must be tested, the workload is very huge, especially for some cultures. Bacteria with strict environmental requirements will increase the workload and difficulty of detection

However, there are also the following disadvantages: 1) Low throughput: only one pathogenic component can be detected at a time. If multiple pathogenic bacteria need to be detected, multiple PCR instruments are required to work at the same time, with low efficiency and long cycle, which will pollute large quantities of multiple pathogens. 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

[0016] At present, there are no relevant research reports on the simultaneous detection of fifteen hemorrhagic fever pathogens and their detection methods based on the GeXP multiple gene expression genetic analysis system at home and abroad

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